Method for determining the amount of an analyte in a sample

ABSTRACT

An automated analyzer for performing multiple diagnostic assays simultaneously includes multiple stations in which discrete aspects of the assay are performed on fluid samples contained in sample vessels. The analyzer includes stations for automatically preparing a sample, incubating the sample, preforming an analyte isolation procedure, ascertaining the presence of a target analyte, and analyzing the amount of a target analyte. An automated receptacle transporting system moves the sample vessels from one station to the next. A method for performing an automated diagnostic assay includes an automated process for isolating and amplifying a target analyte, and, in one embodiment, a method for real-time monitoring of the amplification process.

PRIORITY CLAIM

This application claims the benefit of U.S. Provisional Application No.60/659,874 filed Mar. 10, 2005, the contents of which are herebyincorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates generally to an automated analyzer forsimultaneously performing multiple nucleic acid-based assays, and morespecifically to a system and method for performing multiple nucleic acidamplification assays, including both real-time and end-pointamplifications assays. The present invention also relates to anapparatus and method for continuously processing the contents of aplurality of reaction receptacles following a real-time amplificationprocedure. The present invention further relates to a method forreducing the presence of amplification inhibitors in reactionreceptacles prior to performing nucleic acid amplification reactions.

BACKGROUND OF THE INVENTION

Nucleic acid-based assays can enable highly specific and sensitivedetection of nucleic acid analytes from a variety of sources, includingclinical, industrial, environmental, and food sources. These assays canbe used to determine or monitor for the presence or amount of biologicalantigens (e.g., prions), cell abnormalities, disease states, anddisease-associated pathogens, including parasites, fungi, bacteria andviruses present in a host organism or sample. Nucleic acid-based assaysmay be qualitative or quantitative, with the quantitative assaysproviding useful information to practitioners for evaluating the extentof infection or disease or to determine the state of a disease overtime. Quantitative assays can also be used, for example, to assess theeffectiveness of a therapeutic treatment program or, alternatively, todetermine the extent of an infection or contamination by a particularorganism or virus.

All nucleic acid-based assay formats involve a number of process stepsleading to the identification, detection or quantification of one ormultiple target nucleic acids in a sample. When necessary, thespecifically targeted nucleic acid sequences of a nucleic acid-basedassay may be unique to an identifiable group of organisms (as usedherein, the term “organisms” is inclusive of viruses), where the groupis defined by at least one shared nucleic acid sequence that is commonto all members of the group and that is specific to that group. (A“group” of organisms is generally a phylogenetic grouping of organisms,such as a strain, species or genus of organisms and may be limited to asingle organism.) Generally, the uniqueness of the targeted nucleic acidsequence or sequences need only be limited to the particular sample typebeing assayed (e.g., a human sample versus an industrial orenvironmental sample). Nucleic acid-based methods and means fordetecting individual and groups of organisms are disclosed by Kohne,“Method for Detection, Identification and Quantitation of Non-ViralOrganisms,” U.S. Pat. No. 4,851,330, and Hogan et al., “Nucleic AcidProbes for Detection and/or Quantitation of Non-Viral Organisms,” U.S.Pat. No. 5,541,308.

After determining what organisms are to be targeted by an assay, thefirst step is to select or design a probe which exhibits specificity fora nucleic acid sequence belonging to those organisms which define thegroup. Nucleic acid-based assays can be designed to detect eitherdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA), includingribosomal RNA (rRNA), transfer RNA (tRNA) or messenger RNA (mRNA). Forprokaryotic and eukaryotic organisms, rRNA or the encoding DNA (rDNA) isgenerally a preferred target for detection. Ribosomal RNA sequences areparticularly preferred targets for non-amplified, nucleic acid-basedassays because of their relative abundance in cells, and because rRNAcontains regions of sequence variability that can be exploited to designprobes capable of distinguishing between even closely related organisms.Viruses, which do not contain ribosomal nucleic acid, and cellularchanges are often best detected by targeting DNA, RNA, or a messengerRNA (mRNA) sequence. See, e.g., McDonough et al., “Detection of HumanImmunodeficiency Virus Type 1, U.S. Pat. No. 6,649,749; and Fradet etal., “Methods to Detect Prostate Cancer in a Sample,” U.S. PatentApplication Publication No. US 2005-0282170 A1. Such viruses may includepositive-strand RNA viruses (e.g., hepatitis C virus), where the RNAgenome is mRNA, negative-strand RNA viruses (e.g., influenza viruses),retroviruses (e.g., human immunodeficiency virus), single-stranded DNAviruses (e.g., parvoviruses), and double-stranded DNA viruses (e.g.,adenoviruses), which would require a melting step to render thedouble-stranded target region sufficiently single-stranded foramplification or detection. When the focus of a nucleic acid-based assayis the detection of a genetic abnormality, then the probes are usuallydesigned to detect identifiable changes in the genetic code, an exampleof which is the abnormal Philadelphia chromosome associated with chronicmyelocytic leukemia. See, e.g., Stephenson et al., “Deoxynucleic AcidMolecules Useful as Probes for Detecting Oncogenes Incorporated IntoChromosomal DNA,” U.S. Pat. No. 4,681,840.

When performing a nucleic acid-based assay, preparation of the sample isnecessary to release and stabilize target nucleic acids which may bepresent in the sample. Sample preparation can also serve to eliminatenuclease activity and remove or inactivate potential inhibitors ofnucleic acid amplification (discussed below) or detection of the targetnucleic acids. See, e.g., Ryder et al., “Amplification of Nucleic AcidsFrom Mononuclear Cells Using Iron Complexing and Other Agents,” U.S.Pat. No. 5,639,599, which discloses methods for preparing nucleic acidfor amplification, including the use of complexing agents able tocomplex with ferric ions released by lysed red blood cells. The methodof sample preparation can vary and will depend in part on the nature ofthe sample being processed (e.g., blood, urine, stool, pus or sputum).When target nucleic acids are being extracted from a white blood cellpopulation present in a diluted or undiluted whole blood sample, adifferential lysis procedure is generally followed. See, e.g., Ryder etal., “Preparation of Nucleic Acid From Blood,” European PatentApplication No. 0 547 267 A2. Differential lysis procedures are wellknown in the art and are designed to specifically isolate nucleic acidsfrom white blood cells, while limiting or eliminating the presence oractivity of red blood cell products, such as heme, which can interferewith nucleic acid amplification or detection. Other lytic methods aredisclosed by, for example, Cummins et al., “Methods of ExtractingNucleic Acids and PCR Amplification Without Using a Proteolytic Enzyme,”U.S. Pat. No. 5,231,015, and Clark et al., “Methods for ExtractingNucleic Acids From a Wide Range of Organisms by NonlyticPermeabilization,” U.S. Pat. No. 5,837,452; and Cunningham et al.,“Compositions, Methods and Kits for Determining the Presence ofCryptosporidium Organisms in a Test Sample,” U.S. Patent ApplicationPublication No. US 2002-0055116 A1.

To purify the sample and remove nucleases and other materials capable ofinterfering with amplification or detection, the targeted nucleic acidcan be isolated by target-capture means using a “capture probe” whichbinds the target nucleic acid and is or becomes either directly orindirectly bound to a solid substrate, such as a magnetic or silicaparticle. See, e.g., Ranki et al., “Detection of Microbial Nucleic Acidsby a One-Step Sandwich Hybridization Test,” U.S. Pat. No. 4,486,539;Stabinsky, “Methods and Kits for Performing Nucleic Acid HybridizationAssays,” U.S. Pat. No. 4,751,177; Boom et al., “Process for IsolatingNucleic Acid,” U.S. Pat. No. 5,234,809; Englehardt et al., “CaptureSandwich Hybridization Method and Composition,” U.S. Pat. No. 5,288,609;Collins, “Target and Background Capture Methods and Apparatus forAffinity Assays,” U.S. Pat. No. 5,780,224; and Weisburg et al.,“Two-Step Hybridization and Capture of a Polynucleotide,” U.S. Pat. No.6,534,273. When the solid support is a magnetic particle, magnets inclose proximity to the reaction receptacle are used to draw and hold themagnetic particles to the side of the receptacle, thereby isolating anybound nucleic acid within the reaction receptacle. Other methods forisolating bound nucleic acid in a reaction receptacle includecentrifugation and immobilizing the capture probe on the reactionreceptacle. See, e.g., Boom et al., supra, and Urdea, “PolynucleotideCapture Assay Employing in Vitro Amplification,” U.S. Pat. No.5,200,314. Once the bound nucleic acid is thus isolated, the boundnucleic acid can be separated from unbound nucleic acid and othercellular and sampel material by aspiring the fluid contents of thereaction receptacle and optionally performing one or more wash stepswith a wash solution.

In most cases, it is desirable to amplify the target sequence. Nucleicacid amplification involves the use of nucleic acid polymerases toenzymatically synthesize nucleic acid amplification products (copies)containing a sequence that is either complementary or homologous to thetemplate nucleic acid sequence being amplified. The amplificationproducts may be either extension products or transcripts generated in atranscription-based amplification procedure. Examples of nucleic acidamplification procedures practiced in the art include the polymerasechain reaction (PCR), strand displacement amplification (SDA),loop-mediated isothermal amplification (LAMP) ligase chain reaction(LCR), immuno-amplification, and a variety of transcription-basedamplification procedures, including transcription-mediated amplification(TMA), nucleic acid sequence based amplification (NASBA), andself-sustained sequence replication (3SR). See, e.g., Mullis, “Processfor Amplifying, Detecting, and/or Cloning Nucleic Acid Sequences,” U.S.Pat. No. 4,683,195; Walker, “Strand Displacement Amplification,” U.S.Pat. No. 5,455,166; Notomi et al., “Process for Synthesizing NucleicAcid,” U.S. Pat. No. 6,410,278; Birkenmeyer, “Amplification of TargetNucleic Acids Using Gap Filling Ligase Chain Reaction,” U.S. Pat. No.5,427,930; Cashman, “Blocked-Polymerase Polynucleotide ImmunoassayMethod and Kit,” U.S. Pat. No. 5,849,478; Kacian et al., “Nucleic AcidSequence Amplification Methods,” U.S. Pat. No. 5,399,491; Malek et al.,“Enhanced Nucleic Acid Amplification Process,” U.S. Pat. No. 5,130,238;and Lizardi et al., Bio Technology, 6:1197 (1988). Nucleic acidamplification is especially beneficial when the amount of targetsequence present in a sample is very low. By amplifying the targetsequences and detecting the synthesized amplification product, thesensitivity of an assay can be vastly improved, since fewer targetsequences are needed at the beginning of the assay to ensure detectionof the targeted nucleic acid sequences.

Detection of a target nucleic acid requires the use of a probe having anucleotide base sequence which binds to a target sequence containedwithin the target nucleic acid or, alternatively, amplification productcontaining the target sequence or its complement. Probes useful fordistinguishing between sources of nucleic acid are selected or designedsuch that they do not detectably bind to nucleic acid from non-targetorganisms which may be present in the sample under the selected assayconditions. While probes may include non-nucleotide components, thetarget binding portion of a probe will include DNA, RNA and/or analogsthereof in order to effect hybridization to the target sequence or itscomplement. See, e.g., Becker et al., “Modified Oligonucleotides forDetermining the Presence of a Nucleic Acid Analyte in a Sample,” U.S.Patent Application No. US 2003-0036058 A1 (discloses the use of2′-O-methyl modified probes); and Nielsen et al., “Peptide NucleicAcids,” U.S. Pat. No. 5,539,082 (discloses the use of probes having a2-aminoethylglycine backbone which couples the nucleobase subunits bymeans of a carboxylmethyl linker to the central secondary amine). Fordetection purposes, probes may include a detectable label, such as aradiolabel, fluorescent dye, biotin, enzyme or chemiluminescentcompound, where the label may be provided either before, during or afterhybridization to the probe to the target sequence or its complement.See, e.g., Higuchi, “Homogenous Methods for Nucleic Amplifications andDetection,” U.S. Pat. No. 5,994,056 (discloses the use of intercalatingagents such as eithidium bromide); and Urdea et al., “Solution PhaseNucleic Acid Sandwich Assays Having Reduced Background Noise,” U.S. Pat.No. 5,635,352 (discloses use of label probes for binding to cruciformstructures containing a target nucleic acid).

Nucleic acid-based assays may be based on a homogenous or a heterogenousformat. One form of a heterogenous assay involves preferentially bindinga probe:target complex to a solid support, such as glass, minerals orpolymeric materials, and removing any unbound probe prior to detection.In an alternative approach, it is the unbound probe which is associatedwith the solid support while probe complexed with the target sequenceremains free in solution and can be separated for detection. Homogenousassays generally take place in solution without a solid phase separationstep and commonly exploit chemical differences between a probe free insolution and a probe which has formed part of a target:probe complex. Anexample of a homogenous assay is the Hybridization Protection Assay(HPA), the particulars of which are disclosed by Arnold et al.,“Homogenous Protection Assay,” U.S. Pat. No. 5,639,604. Detection in HPAis based on differential hydrolysis which permits specific detection ofan acridinium ester-labeled probe hybridized to the target sequence orits complement. See, e.g., Arnold et al., “Protected ChemiluminescentLabels,” U.S. Pat. No. 4,950,613; Campbell et al., “ChemilunescentAcridium Labelling Compounds,” U.S. Pat. No. 4,946,958; Arnold et al.,“Acridinium Ester Labelling and Purification of Nucleotide Probes,” U.S.Pat. No. 5,185,439; and Arnold et al., “Linking Reagents for NucleotideProbes,” U.S. Pat. No. 5,585,481. This detection format includes both ahybridization step and a selection step. In the hybridization step, anexcess of acridinium ester-labeled probe is added to the reactionreceptacle and permitted to anneal to the target sequence or itscomplement. Following the hybridization step, label associated withunhybridized probe is rendered non-chemiluminescent in the selectionstep by the addition of an alkaline reagent. The alkaline reagentspecifically hydrolyzes only that acridinium ester label associated withunhybridized probe, leaving the acridinium ester of the probe:targethybrid intact and detectable. Chemiluminescence from the acridiniumester of the hybridized probe can then be measured using a luminometerand signal is expressed in relative light units or RLU.

Other homogenous assays include those disclosed by the following:Gelfand et al., “Reaction Mixtures for Detection of Target NucleicAcids,” U.S. Pat. No. 5,804,375; Nadeau et al., “Detection of NucleicAcids by Fluorescence Quenching,” U.S. Pat. No. 5,958,700; Tyagi et al.,“Detectably Labeled Dual Conformation Oligonucleotide Probes, Assays andKits,” U.S. Pat. No. 5,925,517; Morrison, “Competitive HomogenousAssay,” U.S. Pat. No. 5,928,862; and Becker et al., “Molecular Torches,”U.S. Pat. No. 6,849,412. These patents each describe unimolecular orbimolecular probes which may be used to determine the amount of a targetnucleic acid in an amplification procedure in real-time, where signalchanges associated with the formation of probe:target complexes aredetected during amplification and used to calculate an estimated amountof a target nucleic acid present in a sample. Algorithms for calculatingthe quantity of target nucleic acid originally present in a sample basedon signal information collected during an amplification procedureinclude that disclosed by Wittwer et al., “PCR Method for Nucleic AcidQuantification Utilizing Second or Third Order Rate Constants,” U.S.Pat. No. 6,232,079; Sagner et al., “Method for the Efficiency-CorrectedReal-Time Quantification of Nucleic Acids,” U.S. Pat. No. 6,691,041;McMillan et al., “Methods for Quantitative Analysis of a Nucleic AcidAmplification Reaction,” U.S. Pat. No. 6,911,327; and Chismar et al.,“Method and Algorithm for Quantifying Polynucleotides,” U.S. ProvisionalApplication No. 60/693,455, which enjoys common ownership herewith.

After the nucleic acid-based assay is run, and to avoid possiblecontamination of subsequent amplification reactions, the reactionmixture can be treated with a deactivating reagent which destroysnucleic acids and related amplification products in the reactionreceptacle. Such reagents can include oxidants, reductants and reactivechemicals which modify the primary chemical structure of a nucleic acid.These reagents operate by rendering nucleic acids inert towards anamplification reaction, whether the nucleic acid is RNA or DNA. Examplesof such chemical agents include solutions of sodium hypochlorite(bleach), solutions of potassium permanganate, formic acid, hydrazine,dimethyl sulfate and similar compounds. More details of deactivationprotocols can be found in Dattagupta et al., “Method and Kit forDestroying the Ability of Nucleic Acid to Be Amplified,” U.S. Pat. No.5,612,200, and Nelson et al., “Reagents, Methods and Kits for Use inDeactivating Nucleic Acids,” U.S. Patent Application Publication No. US2005-0202491 A1.

Given the large number of complex steps associated with nucleicacid-based amplification assays, and the different processing andequipment requirements of each type of amplification assay, a needexists for an automated system capable of processing the contents of aplurality of reaction receptacles according to different amplificationassay protocols, and most especially for performing both real-time andend-point amplification assays on the same platform and/or within aself-contained housing. Real-time amplification assays involveperiodically determining the amount of targeted amplification productsas the amplification reaction is taking place, thereby making it easierto provide quantitative information about target nucleic acids presentin a sample, whereas end-point amplifications determine the amount oftargeted amplification products after the amplification reaction hasoccurred, generally making them more useful for providing qualitativeinformation about target nucleic acids. To improve flow through and,thereby, to reduce the amount of time needed to process large volumes ofsamples, there is also a need for a system capable of continuouslyprocessing the contents of multiple reaction receptacles according to areal-time amplification protocol without having to interrupt the systemto manually or automatically load a new batch of reaction receptaclesfor processing. Additionally, there is a need for a reagent and methodfor reducing the amount of amplification inhibitors in reactionreceptacles that could affect qualitative or quantitativedeterminations.

SUMMARY OF THE INVENTION

The above-described needs are addressed by an automated analyzerconstructed and operated in accordance with aspects of the presentinvention. In general, the automated analyzer integrates and coordinatesthe operation of various automated stations, or modules, involved inperforming one or more assays on a plurality of reaction mixturescontained in reaction receptacles. The analyzer is preferably aself-contained, stand alone unit. Assay sample materials and reactionreceptacles, as well as the various solutions, reagents, and othermaterials used in performing the assays are preferably stored within theanalyzer, as are the waste products generated when assays are performed.The analyzer is an integrated nucleic acid testing system that fullyautomates all assay steps from sample processing through amplificationand multi-format detection. In a preferred embodiment, the instrument iscapable of running both real-time and end-point amplification assays.After daily set up is completed, the operator may choose to run anend-point amplification assay, a real-time amplification assay, or both.For real-time amplification assays, the analyzer of the presentinvention is capable of processing the contents of multiple reactionreceptacles in a continuous as opposed to a batch mode, thereby greatlyincreasing the speed at which results can be calculated and reported. Inoperation, the analyzer can also be used to reduce the presence ofamplification inhibitors by providing a surface treating agent that isused to coat the inner surfaces of reaction receptacles prior to orduring an isolation and purification step to remove sample materialand/or reagents from reaction receptacles.

The analyzer includes a computer controller which runsanalyzer-controlling and assay-scheduling software to coordinateoperation of the stations of the analyzer and movement of each reactionreceptacle through the analyzer.

Reaction receptacles can be loaded in an input queue which sequentiallypresents each receptacle at a pick-up position to be retrieved by atransport mechanism, which automatically transports the reactionreceptacles between the stations of the analyzer.

Sample containers are carried on a first ring assembly, and disposablepipette tips are carried on a second ring assembly. Containers of targetcapture reagent, including a suspension of solid support material, arecarried on an inner rotatable assembly constructed and arranged toselectively agitate the containers or present the containers for accessby the probe of an automatic robotic pipette system. Reaction mixtures,including fluid sample material and target capture reagent, are preparedby the pipette system within each reaction receptacle.

The analyzer further includes receptacle mixers for mixing the contentsof a receptacle placed therein. The mixer may be in fluid communicationwith fluid containers and may include dispensers for dispensing one ormore fluids into the receptacle. One or more incubators carry multiplereceptacles in a temperature-controlled chamber and permit individualreceptacles to be automatically placed into and removed from thechamber. Magnetic separation stations automatically perform a magneticseparation and wash procedure on the contents of a receptacle placed inthe station.

In the preferred method of operation, assay results may be ascertainedby the amount of light emitted from a receptacle at the conclusion ofthe appropriate preparation steps. Accordingly, the analyzer includes aluminometer (a type of signal detecting device) for detecting and/orquantifying the amount of light emitted by the contents of the reactionreceptacle. A deactivation queue may be provided to deactivate thecontents of a reaction receptacle placed therein at the conclusion ofthe assay.

Reaction receptacles can be independently transported between stationsby the transport mechanism, and the stations can be operated in parallelto perform different assay procedures simultaneously on differentreaction receptacles, thereby facilitating efficient, high through-putoperation of the analyzer. Moreover, the present invention facilitatesarranging the various stations associated with a nucleic acid-basedassay onto a single, contained platform, thereby achieving efficientspace utilization.

Other objects, features, and characteristics of the present invention,including the methods of operation and the function and interrelation ofthe elements of structure, will become more apparent upon considerationof the following description and the appended claims, with reference tothe accompanying drawings, all of which form a part of this disclosure,wherein like reference numerals designate corresponding parts in thevarious figures.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of an automated nucleic acid-baseddiagnostic analyzer according to the present invention;

FIG. 2 is a perspective view of the structural frame of the analyzer ofthe present invention;

FIG. 3 is a plan view of a portion of the assay processing deck of theanalyzer of the present invention;

FIG. 4 is an exploded perspective view of the assay processing deck;

FIG. 5 is a plan view of a sample ring and a pipette tip wheel of theassay processing deck of the analyzer of the present invention;

FIG. 6 is a perspective view showing the sample ring and the pipette tipwheel;

FIG. 6A is a partial cross-sectional view along the line 6A-6A in FIG.5;

FIG. 7 is a perspective view of a multi-axis mixer of the processingdeck of the analyzer of the present invention;

FIG. 8 is a plan view of the multi-axis mixer;

FIG. 9 is a side elevation of the multi-axis mixer;

FIG. 10 is a plan view of the multi-axis mixer with container holdersand a turntable cover removed therefrom;

FIG. 11 is a cross-sectional view of the multi-axis mixer taken in thedirection 11-11 in FIG. 10;

FIG. 12 is a perspective view of a drive assembly of the multi-axismixer;

FIG. 13 is a perspective view of a transport mechanism of the processingdeck of the analyzer of the present invention;

FIG. 14 is a perspective view of a manipulating hook mounting plate anda manipulating hook actuating mechanism of the transport mechanism, withthe manipulating hook member engaged with a reaction receptacle and in aretracted position;

FIG. 15 is the same as FIG. 14, except with the manipulating hook memberin the extended position;

FIG. 16 is an exploded perspective view of the transport mechanism;

FIG. 17 is a side-elevation of a temperature ramping station of theprocessing deck of the analyzer of the present invention;

FIG. 18 is a front-elevation of the temperature ramping station;

FIG. 19 is a perspective view of a rotary incubator of the processingdeck of the analyzer of the present invention;

FIG. 20 is an exploded view of a portion of a housing and access openingclosure mechanisms according to a first embodiment of the rotaryincubator;

FIG. 21 is a partial view of a skewed disk linear mixer of the rotaryincubator, shown engaged with a reaction receptacle employed in apreferred mode of operation of the analyzer of the present invention;

FIG. 22 is an exploded perspective view of the first embodiment of therotary incubator;

FIG. 23 is a perspective view of the rotary incubator according to asecond embodiment thereof;

FIG. 23A is an exploded perspective view of the second embodiment of therotary incubator;

FIG. 23B is a partial exploded perspective view of an access openingclosure mechanism of the second embodiment of the rotary incubator;

FIG. 23C is an exploded view of a receptacle carrier carousel of thesecond embodiment of the rotary incubator;

FIG. 24 is a perspective view of a particles of the processing deck ofthe present invention with a side plate thereof removed;

FIG. 25 is a partial transverse cross-section of the particles;

FIG. 25A is a partial transverse cross-section of a tip of an aspiratingtube of the particles with a contamination-limiting tiplet carried onthe end thereof;

FIG. 26 is an exploded perspective view of a receptacle carrier unit, anorbital mixer assembly, and a divider plate of the particles;

FIG. 27 is a partial cross-sectional view of a wash solution dispensernozzle, an aspirator tube with a contamination-limiting tiplet engagedwith an end thereof, and a receptacle carrier unit of the particles,showing a multi-tube unit reaction receptacle employed in a preferredmode of operation of the analyzer carried in the receptacle carrier unitand the aspirator tube and contamination-limiting tiplet inserted into areaction tube of the multi-tube unit;

FIG. 28 is a partial cross-sectional view of the wash solution dispensernozzle, the aspirator tube, and the receptacle carrier unit of theparticles, showing the multi-tube unit carried in the receptacle carrierunit and the aspirator tube engaging the contamination-limiting tipletheld in a contamination-limiting element holding structure of themulti-tube unit;

FIGS. 29A-29D show a partial cross-section of a first embodiment of atiplet stripping hole of a tiplet stripping plate of the particles and atiplet stripping operation using the tiplet stripping hole;

FIGS. 30A-30D show a partial cross-section of a second embodiment of atiplet stripping hole and a tiplet stripping operation using the tipletstripping hole;

FIG. 31A is a plan view of a third embodiment of a tiplet stripping holeof a tiplet stripping plate of the particles;

FIGS. 31B-31C show a partial cross-section of the third embodiment ofthe tiplet stripping hole and a tiplet stripping operation using thetiplet;

FIG. 32 is a perspective view of an orbital mixer with a front platethereof removed;

FIG. 33 is an exploded view of the orbital mixer of the processing deckof the analyzer of the present invention;

FIG. 34 is a top-plan view of the orbital mixer;

FIG. 35 is a top perspective view of a reagent cooling bay of theprocessing deck of the analyzer of the present invention;

FIG. 36 is a top perspective view of a reagent cooling bay with thecontainer tray removed therefrom;

FIG. 37 is a bottom plan view of the reagent cooling bay;

FIG. 38 is an exploded view of the reagent cooling bay;

FIG. 39 is a top perspective view of a modular container tray of thereagent cooling bay;

FIG. 40 is a perspective view of a first embodiment of a luminometer ofthe processing deck of the analyzer of the present invention;

FIG. 41 is a partial exploded perspective view of the luminometer of thefirst embodiment;

FIG. 42A is a partial perspective view of a receptacle transportmechanism of the first embodiment of the luminometer;

FIG. 42B is an end view of the receptacle transport mechanism of thefirst embodiment of the luminometer;

FIG. 42C is a top view of the receptacle transport mechanism of thefirst embodiment of the luminometer;

FIG. 43 is a break away perspective view of a second embodiment of theluminometer of the present invention;

FIG. 44 is an exploded perspective view of a multi-tube unit doorassembly for the luminometer of the second embodiment;

FIG. 45 is an exploded perspective view of a shutter assembly for aphotosensor aperture for the luminometer of the second embodiment;

FIG. 45A is a perspective view of an aperture plate of the shutterassembly of the luminometer of the second embodiment;

FIG. 46 is a perspective view of a reaction tube positioner assembly ofthe luminometer of the second embodiment, including a reaction tubepositioner disposed within a reaction tube positioner frame;

FIG. 47 is a perspective view of the reaction tube positioner;

FIG. 48 is a side elevation of the reaction tube positioner assembly;

FIG. 49 is a perspective view showing the reaction tube positioner ofthe reaction tube positioner assembly operatively engaging a multi-tubeunit employed in a preferred mode of operation of the analyzer;

FIG. 50 is a perspective view of a multi-tube unit transport mechanismof the luminometer of the second embodiment;

FIG. 51 is a partial perspective view showing a multi-tube unittransport and drive screw of the multi-tube unit transport mechanism ofthe luminometer;

FIG. 52 is a perspective view of a lower chassis of the analyzer of thepresent invention;

FIG. 53 is a perspective view of a right-side drawer of the lowerchassis;

FIG. 54 is a perspective view of a left-side drawer of the lowerchassis;

FIG. 55 is a perspective view of a sample tube tray employed in apreferred mode of operation of the analyzer of the present invention;

FIG. 56 is a top plan view of the sample tube tray;

FIG. 57 is a partial cross-section of the sample tube tray through line“57-57” in FIG. 55;

FIG. 58 is a perspective view of a multi-tube unit employed in apreferred mode of operation of the analyzer of the present invention;

FIG. 59 is a side elevation of a contact-limiting pipette tipletemployed in a preferred mode of operation of the analyzer of the presentinvention and carried on the multi-tube unit shown in FIG. 58; and

FIG. 60 is an enlarged bottom view of a portion of the multi-tube unit,viewed in the direction of arrow “60” in FIG. 58.

FIG. 61 is a side elevation in cross-section showing an opticaldetection module and portions of a real-time fluorometer and amulti-tube unit;

FIG. 62 is an exploded perspective view of the housing of the opticaldetection module;

FIG. 63 is an exploded perspective view of the optical detection module;

FIG. 64 is a top plan view of a real-time fluorometer showing preferredpositions of the optical detection modules;

FIG. 65 is a schematic view of a real-time fluorometer showing preferredpositions of the optical detection modules;

FIG. 66 is a graph showing excitation spectra of preferred amplificationdetection dyes;

FIG. 67 is a graph showing emission spectra of preferred amplificationdetection dyes;

FIGS. 68A-68F show a diagram of a circuit for the optical detectionmodule;

FIG. 69 is a perspective view showing the optical detector scanningassembly of a scanning real-time fluorometer;

FIG. 70 is a side elevation of the detector scanning assembly;

FIG. 71 is a top plan view of the detector scanning assembly;

FIG. 72 is a bottom plan view of the detector scanning assembly.

FIG. 73 is a perspective view of a portion of a carousel of thereal-time fluorometer;

FIG. 74 is an exploded perspective view showing the carousel of thereal-time fluorometer and a magnetic divider;

FIG. 75 is a bottom plan view of the carousel of the real-timefluorometer showing a single magnetic divider attached thereto;

FIG. 75A is a partial cross-sectional view taken along the line A-A inFIG. 75;

FIG. 76A is a flow chart showing the protocols of a preferred real-timeamplification assay and a portion of a preferred end-point amplificationassay which stops after exposure to amplification conditions, bothassays being in accordance with the present invention;

FIG. 76B is a flow chart showing the remainder of the protocol for thepreferred end-point amplification assay of FIG. 76A following exposureto amplification conditions;

FIG. 77 is a flow chart showing an analyte quantification process;

FIG. 78 is a time plot of real-time fluorometer data; and

FIG. 79 is a plot showing a method for fitting a curve to real-timefluorometer data and using the fit to determine a threshold time.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

While the present invention may be embodied in a variety of forms, thefollowing descriptions and accompanying drawings are merely intended todisclose some of those forms as specific examples of the presentinvention. Accordingly, the present invention is not intended to belimited to the forms or embodiments so described and illustrated.Instead, the full scope of the present invention is set forth in theappended claims.

Analyzer Overview

An automated diagnostic analyzer according to the present invention isdesignated generally by reference number 50 in FIGS. 1 and 2. Analyzer50 includes a housing 60 built over an internal frame structure 62,preferably made of steel. The analyzer 50 is preferably supported oncaster wheels 64 structurally mounted to the frame structure 62 so as tomake the analyzer movable.

The various stations involved in performing an automated assay and theassay samples are housed within housing 60. In addition, the varioussolutions, reagents, and other materials used in performing the assaysare preferably stored within the housing 60, as are the waste productsgenerated when assays are performed with the analyzer 50.

Housing 60 includes a test receptacle loading opening 68, which is shownin FIG. 1 to be disposed in a forwardly facing panel of the housing 60,but could as well be located in other panels of the housing 60. Apipette door 70 having a view window 72 and a carousel door 74 having aview window 76 are disposed above a generally horizontal work surface66. A forwardly protruding arcuate panel 78 accommodates a samplecarousel, which will be described below. A flip-up arcuate sample door80 is pivotally attached to the housing so as to be vertically pivotalwith respect to arcuate panel 78 so as to provide access to a forwardportion of the sample carousel behind the panel 78. Sensors indicatewhen the doors are closed, and the sample door 80, the carousel door 74,and the pipette door 70 are locked during analyzer operation. Thelocking mechanism for each door preferably consists of a hook attachedto a DC rotary solenoid (rated for continuous duty) with a springreturn. Preferred rotary solenoids are available from Lucas ControlSystems, of Vandalia, Ohio, Model Nos. L-2670-034 and L-1094-034.

An extension portion 102, preferably made of a transparent ortranslucent material, extends above the top portion of housing 60 so asto provide vertical clearance for moving components within the housing60.

The assays are performed primarily on a processing deck 200, which isthe general location of the various assay stations of the analyzer 50described below. For simplicity of the illustration, the processing deck200 is shown in FIG. 2 without any of the assay stations mountedthereon. The processing deck 200 comprises a datum plate 82 to which thevarious stations are directly or indirectly mounted. Datum plate 82preferably comprises a machined aluminum plate. The processing deck 200,also known as the chemistry deck, separates the interior of the housinginto the chemistry area, or upper chassis, above the datum plate 82 andthe storage areas, or lower chassis 1100, located below the datum plate82.

A number of fans and louvers are preferably provided in the upperchassis portion of the housing 60 to create air circulation throughoutthe upper chassis to avoid excessive temperatures in the upper chassis.

As the analyzer 50 of the present invention is computer controlled, theanalyzer 50 includes a computer controller, schematically represented asbox 1000 in FIG. 2, which runs high-level analyzer-controlling softwareknown as the “assay manager program”. The assay manager program includesa scheduler routine which monitors and controls test sample movementthrough the chemistry deck 200.

The computer controller 1000 which controls the analyzer 50 may includea stand-alone computer system including a CPU, keyboard, monitor, andmay optionally include a printer device. A portable cart may also beprovided for storing and supporting the various computer components.Alternately, the computer hardware for running the analyzer-controllingsoftware may be integrally housed within the housing 60 of the analyzer50.

Low level analyzer control, such as control of electric motors andheaters used throughout the analyzer 50 and monitoring of fluid levelswithin bulk fluid and waste fluid containers, is performed by anembedded controller, preferably comprising a Motorola 68332microprocessor. Stepper motors used throughout the analyzer are alsopreferably controlled by preprogrammed, off-the-shelf, microprocessorchips available from E-M Technologies, Bala Cynwyd, Pa.

The processing deck 200 is shown schematically in FIGS. 3 and 4. FIG. 3represents a schematic plan view of a portion of the processing deck200, and FIG. 4 represents a schematic perspective view of theprocessing deck. The datum plate 82 forms the foundation of theprocessing deck 200 on which all stations are directly or indirectlyattached.

Processing deck 200 includes a reaction receptacle input queue 150 whichextends from opening 68 in front of housing 60. A plurality of reactionreceptacles are loaded in a stacked fashion in the input queue 150. Thepurpose of the input queue is to hold a prescribed number of reactionreceptacles and to sequentially present them at a pick-up position to beretrieved by a transport mechanism (described below). A reflectivesensor at the pick-up position verifies the presence of a receptacle atthat position. The input queue also includes a device for counting thenumber of receptacles resident therein at any given time.

A reaction receptacle shuttle assembly (not shown) within the queuemoves the receptacles along a receptacle advance path toward the pick-upposition. Optical sensors indicate when the shuttle assembly is in itshome and fully extended positions. The queue includes a drawer which maybe pulled out for loading the receptacles therein. Before the drawer isopened, however, it must be unlocked and the shuttle must disengage fromthe receptacle advance path. When the drawer is again closed, it islocked and the shuttle engages the receptacles and moves them toward thepick-up position. Optical sensors indicate when the drawer is closed andwhen the shuttle has engaged a receptacle. As each receptacle is removedfrom the pick-up position by the transport mechanism, the receptacleshuttle advances the receptacles one receptacle-width, so that the nextreceptacle is in the pick-up position.

While the analyzer 50 may be adapted for use with reaction receptaclesconsisting of single reaction receptacles or integrally formed unitscontaining a plurality of reaction receptacles having any of a number ofdifferent shapes, sizes and configurations, the reaction receptacles ofthe present invention are preferably integrally formed linear arrays ofreaction tubes known as multi-tube units or MTUs. These preferredreaction receptacles will be described in more detail below.

A first ring assembly, which in the preferred embodiment comprises asample ring 250, is mounted on a pivoting jig plate 130 at a distanceabove the datum plate 82. Sample ring 250 is generally circular andpreferably holds up to nine sample trays 300 in an annular fluidcontainer carrier portion thereof, and each of the sample trayspreferably holds 20 sample-containing containers, or test tubes 320. Thesample ring 250 is constructed and arranged to be rotatable about afirst generally vertical axis of rotation and delivers the sample tubes320 to a sample pipette assembly 450, preferably an automated roboticpipette system. The forward portion of sample ring 250 is accessiblethrough the flip-up carousel door 80 provided in housing 60 so thattrays 300 of test tubes 320 can be easily loaded onto the sample ring250 and unloaded from the sample ring. Sample ring 250 is driven by amotor, as will be described in more detail below.

A second ring assembly, which in the preferred embodiment comprises apipette tip wheel 350, is located in an interior portion of the samplering 250, so that at least a portion of the outer perimeter of thepipette tip wheel 350 is disposed radially inwardly of the innerperiphery of the ring 250. Pipette tip wheel 350 carries thereon aplurality of commercially available packages of pipette tips. Pipettetip wheel 350 is motor driven to rotate independently of sample ring 250about a second axis of rotation that is generally parallel to the firstaxis of rotation of the sample ring 250.

An inner rotatable assembly constructed and arranged to carry aplurality of fluid containers is provided at an interior portion of thepipette tip wheel 350. In the preferred embodiment, the inner rotatableassembly comprises a multi-axis mixer 400 located radially inside thepipette tip wheel 350 (i.e., the second ring assembly) and sample ring250 (i.e., the first ring assembly). The multi-axis mixer 400 includes arotating turntable 414 that is rotatable about a third axis of rotationthat is generally parallel to the first and second axes of rotation andon which are mounted four independently and eccentrically rotatingcontainer holders 406. Each of the container holders 406 receives acontainer, preferably in the form of a plastic bottle, containing afluid suspension of magnetic particles with immobilized polynucleotidesand polynucleotide capture probes. Each container holder 406 isgenerally cylindrical in shape and includes an axis of symmetry, or axisof rotation. The multi-axis mixer 400 rotates each of the containerseccentrically with respect to the center of the holder 406, whilesimultaneously rotating the turntable 414 about its center so as toprovide substantially constant agitation of the containers to maintainthe magnetic particles in suspension within the fluid.

The sample pipette assembly, or robot, 450 is mounted to the framestructure 62 (see FIG. 2) in a position above the sample ring 250 andpipette tip wheel 350. The sample pipette assembly 450 includes apipette unit 456 having a tubular probe 457 mounted on a gantry assemblyto provide X, Y, Z motion. Specifically, the pipette unit 456 islinearly movable in the Y-direction along a track 458 formed in alateral rail 454, and the lateral rail 454 is longitudinally movable inthe X-direction along a longitudinal track 452. The pipette unit 456provides vertical, or Z-axis motion of the probe 457. Drive mechanismswithin the sample pipette assembly 450 position the pipette unit 456 tothe correct X, Y, Z coordinates within the analyzer 50 to pipettefluids, to wash the probe 457 of the pipette unit 456, to discard aprotective tip from an end of the probe 457 of the pipette unit 456, orto stow the pipette unit 456 during periods of nonuse, e.g., in a “home”position. Each axis of the sample pipette assembly 450 is driven by astepper motor in a known and conventional manner.

The pipette assembly is preferably an off-the-shelf product. Presentlypreferred is the Robotic Sample Processor, Model No. RSP9000, availablefrom Cavro Inc. of Sunnyvale, Calif. This model includes a single gantryarm.

The sample pipette assembly 450 is preferably coupled to a syringe pump(not shown) (the Cavro XP 3000 has been used) and a DC driven diaphragmsystem fluid wash pump (not shown). The syringe pump of the samplepipette assembly 450 is preferably mounted to the internal framestructure 62 within the housing 60 of the analyzer 50 at a positionabove the left-hand side of the chemistry deck 200 and is connected topipette unit 456 by suitable tubing (not shown) or other conduitstructures.

A sample preparation opening 252 is provided in the jig plate 130, sothat the sample pipette assembly 450 can access a reaction receptacle160 in the input queue 150 located below the jig plate 130.

The sample pipette assembly 450 of the analyzer 50 engages sample tubes320 carried on the sample ring 250 through openings 140, 142 of anelevated cover plate 138 and engages pipette tips carried on the pipettetip wheel 350 near the back portions of the sample ring 250 and pipettetip wheel 350, respectively. Accordingly, an operator can have access tothe forward portions of sample ring 250 and pipette tip wheel 350through the carousel door opening 80 during operation of the analyzerwithout interfering with pipetting procedures.

A tip wash/disposal station 340 is disposed adjacent to the sample ring250 on the jig plate 130. Station 340 includes a tip disposal tube 342and a wash station basin 346. During sample preparation, the pipetteunit 456 of the sample pipette assembly 450 can move into position abovethe wash station basin 346 where the tubular probe 457 can be washed bypumping distilled water through the probe 457, the basin of the washstation 346 being connected, preferably by a flexible hose (not shown),to a liquid waste container in the lower chassis 1100.

The tip disposal tube 342 comprises an upstanding tubular member. Duringsample transfer from a sample tube 320 to a reaction receptacle 160, anelongated pipette tip is frictionally secured onto the end of thetubular probe 457 of the pipette unit 456, so that sample material doesnot come into contact with the tubular probe 457 of the pipette unit 456when material is drawn from a sample tube 320 and into the elongatedpipette tip. After a sample has been transferred from a sample tube 320,it is critical that the pipette tip used in transferring that sample notbe used again for another unrelated sample. Therefore, after sampletransfer, the pipette unit 456 moves to a position above the tipdisposal tube 342 and ejects the used, disposable pipette tip into thetip disposal tube 342 which is connected to one of the solid wastecontainers carried in the lower chassis 1100.

An elongated pipette tip is preferably also frictionally secured to theprobe 457 for transferring target capture reagent from containerscarried on the multi-axis mixer 400 to a reaction receptacle 160.Following reagent transfer, the pipette tip is discarded.

As noted, the sample ring 250, the pipette tip wheel 350, and themulti-axis mixer 400 are preferably mounted on a hinged jig plate 130(see FIGS. 5 and 6) supported above the datum plate 82. The jig plate130 is hinged at a back end 132 thereof (see FIG. 6) so that the plate,and the ring 250, the wheel 350, and the mixer 400 mounted thereon, canbe pivoted upwardly to permit access to the area of the chemistry deckbelow the jig plate.

A first, or right-side, transport mechanism 500 is mounted on the datumplate 82 below the jig plate 130 and sample ring 250 on generally thesame plane as the input queue 150. Transport mechanism 500 includes arotating main body portion 504 defining a receptacle carrier assemblyand an extendible manipulating hook 506 mounted within the main body 504and extendible and retractable with respect thereto by means of apowered hook member drive assembly. Each of the reaction receptacles 160preferably includes manipulating structure that can be engaged by theextendible manipulating hook 506, so that the transport mechanism 500can engage and manipulate a reaction receptacle 160 and move it from onelocation on the processing deck 200 to another as the reactionreceptacle is sequentially moved from one station to another during theperformance of an assay within the reaction receptacle 160.

A second, or left-side, transport mechanism 502, of substantiallyidentical construction as first transport mechanism 500, is alsoincluded on the processing deck 200.

A plurality of receptacle parking stations 210 are also located belowthe jig plate 130. The parking stations 210, as their name implies, arestructures for holding sample-containing reaction receptacles until theassay performing stations of the processing deck 200 of the analyzer 50are ready to accept the reaction receptacles. The reaction receptaclesare retrieved from and inserted into the parking stations 210 asnecessary by the transport mechanism 500.

A right-side orbital mixer 550 is attached to the datum plate 82 andreceives reaction receptacles 160 inserted therein by the right-sidetransport mechanism 500. The orbital mixer is provided to mix thecontents of the reaction receptacle 160. After mixing is complete, theright-side transport mechanism 500 removes the reaction receptacle fromthe right-side orbital mixer 550 and moves it to another location in theprocessing deck.

A number of incubators 600, 602, 604, 606, of substantially identicalconstruction are provided. Incubators 600, 602, 604, and 606 arepreferably rotary incubators. Although the particular assay to beperformed and the desired throughput will determine the desired numberof necessary incubators, four incubators are preferably provided in theanalyzer 50.

As will be described in more detail below, each incubator (600, 602,604, 606) has a first, and may also have a second, receptacle accessopening through which a transport mechanism 500 or 502 can insert areaction receptacle 160 into the incubator or retrieve a reactionreceptacle 160 from the incubator. Within each incubator (600, 602, 604,606) is a rotating receptacle carrier carousel which holds a pluralityof reaction receptacles 160 within individual receptacle stations whilethe receptacles are being incubated. For the nucleic acid-baseddiagnostic assay preferably performed on the analyzer 50 of the presentinvention, first rotary incubator TC is a TC incubator (also known asthe “TC incubator”), second rotary incubator 602 is an activetemperature and pre-read cool-down incubator (also known as the “ATincubator”), third rotary incubator 604 is an amplification incubator(also known as the “AMP incubator”), and fourth rotary incubator 606 isa hybridization incubator (also known as the “HYB incubator”). (Thenames assigned to the incubators are for convenience only, signifyingtheir uses in one preferred end-point amplification assay, and are notto be viewed as limiting other possible uses of these incubators). Theconstruction, function, and role of the incubators in the overallperformance of the assay will be described in more detail below.

The processing deck 200 preferably also includes a plurality oftemperature ramping stations 700. Two such stations 700 are shownattached to the datum plate 82 between incubators 602 and 604 in FIG. 3.Additional ramping stations may be disposed at other locations on theprocessing deck 200 where they will be accessible by one of thetransport mechanisms 500, 502.

A reaction receptacle 160 may be placed into or removed from atemperature ramping station 700 by either transport mechanism 500 or502. Each ramping station 700 either raises or lowers the temperature ofthe reaction receptacle and its contents to a desired temperature beforethe receptacle is placed into an incubator or another temperaturesensitive station. By bringing the reaction receptacle and its contentsto a desired temperature before inserting it into one of the incubators(600, 602, 604, 606), temperature fluctuations within the incubator areminimized.

The processing deck 200 also includes magnetic separation stations 800for performing a magnetic separation wash procedure. Each magneticseparation station 800 can accommodate and perform a wash procedure onone reaction receptacle 160 at a time. Therefore, to achieve the desiredthroughput, five magnetic separation stations 800 working in parallelare preferred. Receptacles 160 are inserted into and removed from themagnetic separation stations 800 by the left-side transport mechanism502.

A reagent cooling bay 900 is attached to the datum plate 82 roughlybetween the incubators 604 and 606. Reagent cooling bay 900 comprises acarousel structure having a plurality of container receptacles forholding bottles of temperature sensitive reagents. The carousel resideswithin a cooled housing structure having a lid with pipette-access holesformed therein.

A second, or left-side, orbital mixer 552, substantially identical toright-side orbital mixer 550, is disposed between incubators 606 and604. The left-side orbital mixer 552 includes dispenser nozzles andlines for dispensing fluids into the reaction receptacle resident withinthe left-side orbital mixer 552.

A reagent pipette assembly, or robot, 470 includes a double gantrystructure attached to the frame structure 62 (see FIG. 2) and isdisposed generally above the incubators 604 and 606 on the left-handside of the processing deck 200. Specifically, reagent pipette assembly470 includes pipette units 480 and 482. Pipette unit 480 includes atubular probe 481 and is mounted for linear movement, generally in theX-direction, along track 474 of lateral rail 476, and pipette unit 482,including a tubular probe 483, is also mounted for linear motion,generally in the X-direction, along track 484 of lateral rail 478.Lateral rails 476 and 478 can translate, generally in a Y-direction,along the longitudinal track 472. Each pipette unit 480, 482 providesindependent vertical, or Z-axis, motion of the respective probe 481,483. Drive mechanisms within the assembly 470 position the pipette units480, 482 to the correct X, Y, Z coordinates within the analyzer 50 topipette fluids, to wash the tubular probes 481, 483 of the respectivepipette units 480, 482, or to stow the pipette units 480, 482 duringperiods of nonuse, e.g., in “home” positions. Each axis of the pipetteassembly 470 is driven by a stepper motor.

The reagent pipette assembly 470 is preferably an off-the-shelf product.The presently preferred unit is the Cavro Robotic Sample Processor,Model No. RSP9000, with two gantry arms.

The pipette units 480, 482 of the reagent pipette assembly 470 are eachpreferably coupled to a respective syringe pump (not shown) (the CavroXP 3000 has been used) and a DC driven diaphragm system fluid wash pump.The syringe pumps of the reagent pipette assembly 470 are preferablymounted to the internal frame structure 62 within the housing 60 of theanalyzer 50 at a position above the left-hand side of the chemistry deck200 and are connected to the respective pipette units 480, 482 bysuitable tubing (not shown) or other conduit structures.

Each pipette unit 480, 482 preferably includes capacitive level sensingcapability. Capacitive level sensing, which is generally known in themedical instrumentation arts, employs capacitance changes when thedielectric of a capacitor, formed by the pipette unit as one plate ofthe capacitor and the structure and hardware surrounding a containerengaged by the pipette unit as the opposite plate, changes from air tofluid to sense when the probe of the pipette unit has penetrated fluidwithin a container. By ascertaining the vertical position of the probeof the pipette unit, which may be known by monitoring the stepper motorwhich drives vertical movement of the pipette unit, the level of thefluid within the container engaged by the pipette unit may bedetermined.

Pipette unit 480 transfers reagents from the reagent cooling bay 900into reaction receptacles disposed within the HYB incubator 606 or theorbital mixer 552, and pipette unit 482 transfers reagent materials fromthe reagent cooling bay 900 into reaction receptacles disposed withinthe AMP incubator 604 or the orbital mixer 552.

The pipette units 480, 482 use capacitive level sensing to ascertainfluid level within a container and submerge only a small portion of theend of the probe of the pipette unit to pipette fluid from thecontainer. Pipette units 480, 482 preferably descend as fluid ispipetted into the respective tubular probes 481, 483 to keep the end ofthe probes submerged to a constant depth. After drawing reagent into thetubular probe of the pipette unit 480 or 482, the pipette units create aminimum travel air gap of 10 μl in the end of the respective probe 481or 483 to ensure no drips from the end of the probe as the pipette unitis moved to another location above the chemistry deck 200.

The results of the assay preferably performed in the analyzer 50 of thepresent invention are ascertained by the amount of chemiluminescence, orlight, emitted from a reaction tube 162 at the conclusion of theappropriate preparation steps. Specifically, the results of the assayare determined from the amount of light emitted by label associated withhybridized polynucleotide probe at the conclusion of the assay.Accordingly, the processing deck 200 includes a luminometer 950 fordetecting and/or quantifying the amount of light emitted by the contentsof the reaction receptacle. Briefly, the luminometer 950 comprises ahousing through which a reaction receptacle travels under the influenceof a transport mechanism, a photomultiplier tube, and associatedelectronics. Various luminometer embodiments will be described in detailbelow.

The processing deck 200 also preferably includes a deactivation queue750. The assay performed in the analyzer 50 involves the isolation andamplification of nucleic acids belonging to at least one organism orcell of interest. Therefore, it is desirable to deactivate the contentsof the reaction receptacle 160, typically by dispensing a bleach-basedreagent into the reaction receptacle 160 at the conclusion of the assay.This deactivation occurs within the deactivation queue 750.

Following deactivation, the deactivated contents of the reactionreceptacle 160 are stored in one of the liquid waste containers of thelower chassis 1100 and the used reaction receptacle is discarded into adedicated solid waste container within the lower chassis 1100. Thereaction receptacle is preferably not reused.

Analyzer Operation

The operation of the analyzer 50, and the construction, cooperation, andinteraction of the stations, components, and modules described abovewill be explained by describing the operation of the analyzer 50 on asingle test sample in the performance of one type of assay which may beperformed with analyzer 50. Other diagnostic assays, which require theuse of one or more of the stations, components, and modules describedherein, may also be performed with the analyzer 50. The descriptionherein of a particular assay procedure is merely for the purpose ofillustrating the operation and interaction of the various stations,components, and modules of the analyzer 50 and is not intended to belimiting. Those skilled in the art of diagnostic testing will appreciatethat a variety of chemical and biological assays can be performed in anautomated fashion with the analyzer 50 of the present invention.

The analyzer 50 is initially configured for an assay run by loading bulkfluids into the bulk fluid storage bay of the lower chassis 1100 andconnecting the bulk fluid containers to the appropriate hoses (notshown).

The analyzer is preferably powered up in a sequential process, initiallypowering the stations, or modules, that will be needed early in theprocess, and subsequently powering the stations that will not be neededuntil later in the process. This serves to conserve energy and alsoavoids large power surges that would accompany full analyzer power-upand which could trip circuit breakers. The analyzer also employs a“sleep” mode during periods of nonuse. During sleep mode, a minimalamount of power is supplied to the analyzer, again to avoid large surgesnecessary to power-up an analyzer from complete shut-down.

A number of reaction receptacles 160, preferably in the form of plastic,integrally formed multiple-tube units (MTUs), which are described inmore detail below, are loaded through opening 68 into the input queue150. Henceforth, the reaction receptacles 160 will be referred to asMTUs, consistent with the preferred manner of using the analyzer 50.

The reaction receptacle shuttle assembly (not shown) within the inputqueue 150 moves the MTUs 160 from the loading opening 68 to the pick-upposition at the end of the queue 150. The right-side transport mechanism500 takes an MTU 160 from the end of the queue 150 and moves to a barcode reader (not shown) to read the unique bar code label on that MTUwhich identifies that MTU. From the bar code reader, the MTU is moved toan available sample transfer station 255 below opening 252.

Multiple Tube Units

The preferred MTU is an embodiment of a multi-vessel reaction receptacledisclosed by Horner et al., “Reaction Receptacle Apparatus,” U.S. Pat.No. 6,086,827. As shown in FIG. 58, an MTU 160 comprises a plurality ofindividual reaction tubes 162, preferably five. The reaction tubes 162,preferably in the form of cylindrical tubes with open top ends andclosed bottom ends, are connected to one another by a connecting ribstructure 164 which defines a downwardly facing shoulder extendinglongitudinally along either side of the MTU 160. In one embodiment, thedimensions of each reaction tube 162 of the MTU 160 are 12×75 mm,although the analyzer 50 could be readily adapted to accommodatedifferently dimensioned reaction receptacles which are providedindividually or as part of a multi-vessel reaction receptacle.

The MTU 160 is preferably formed from injection molded polypropylene.The most preferred polypropylene is sold by Montell Polyolefins, ofWilmington, Del., product number PD701NW. The Montell material is usedbecause it is readily moldable, chemically compatible with the preferredmode of operation of the analyzer 50, and has a limited number of staticdischarge events which can interfere with accurate detection orquantification of chemiluminescence.

An arcuate shield structure 169 is provided at one end of the MTU 160.An MTU manipulating structure 166 to be engaged by one of the transportmechanisms 500, 502 extends from the shield structure 169. MTUmanipulating structure 166 comprises a laterally extending plate 168extending from shield structure 169 with a vertically extending piece167 on the opposite end of the plate 168. A gusset wall 165 extendsdownwardly from lateral plate 168 between shield structure 169 andvertical piece 167.

As shown in FIG. 60 the shield structure 169 and vertical piece 167 havemutually facing convex surfaces. The MTU 160 is engaged by the transportmechanisms 500, 502 and other components, as will be described below, bymoving an engaging member laterally (in the direction “A”) into thespace between the shield structure 169 and the vertical piece 167. Theconvex surfaces of the shield structure 169 and vertical piece 167provide for wider points of entry for an engaging member undergoing alateral relative motion into the space. The convex surfaces of thevertical piece 167 and shield structure 169 include raised portions 171,172, respectively, formed at central portions thereof. The purpose ofportions 171, 172 will be described below.

A label-receiving structure 174 having a flat label-receiving surface175 is provided on an end of the MTU 160 opposite the shield structure169 and MTU manipulating structure 166. Labels, such as scannable barcodes, can be placed on the surface 175 to provide identifying andinstructional information on the MTU 160.

The MTU 160 preferably includes tiplet holding structures 176 adjacentthe open mouth of each respective reaction tube 162. Each tiplet holdingstructure 176 provides a cylindrical orifice within which is received acontact-limiting tiplet 170. The construction and function of the tiplet170 will be described below. Each holding structure 176 is constructedand arranged to frictionally receive a tiplet 170 in a manner thatprevents the tiplet 170 from falling out of the holding structure 176when the MTU 160 is inverted, but permits the tiplet 170 to be removedfrom the holding structure 176 when engaged by a pipette.

As shown in FIG. 59, the tiplet 170 comprises a generally cylindricalstructure having a peripheral rim flange 177 and an upper collar 178 ofgenerally larger diameter than a lower portion 179 of the tiplet 170.The tiplet 170 is preferably formed from conductive polypropylene. Whenthe tiplet 170 is inserted into an orifice of a holding structure 176,the flange 177 contacts the top of structure 176 and the collar 178provides a snug but releasable interference fit between the tiplet 170and the holding structure 176.

An axially extending through-hole 180 passes through the tiplet. Hole180 includes an outwardly flared end 181 at the top of the tiplet 170which facilitates insertion of a pipette tubular probe (not shown) intothe tiplet 170. Two annular ridges 183 line the inner wall of hole 180.Ridges 183 provide an interference friction fit between the tiplet 170and a tubular probe inserted into the tiplet 170.

The bottom end of the tiplet 170 preferably includes a beveled portion182. When tiplet 170 is used on the end of an aspirator that is insertedto the bottom of a reaction receptacle, such as a reaction tube 162 ofan MTU 160, the beveled portion 182 prevents a vacuum from formingbetween the end of the tiplet 170 and the bottom of the reactionreaction tube.

Lower Chassis

An embodiment of the lower chassis of the present invention is shown inFIGS. 52-54. The lower chassis 1100 includes a steel frame 1101 with ablack polyurethane powder coat, a pull-out drip tray 1102 disposed belowthe chassis, a right-side drawer 1104, and a left-side drawer 1106. Theleft-side drawer 1106 is actually centrally disposed within the lowerchassis 1100. The far left-side of the lower chassis 1100 houses variouspower supply system components and other analyzer mechanisms such as,for example, seven syringe pumps 1152 mounted on a mounting platform1154, a vacuum pump 1162 preferably mounted on the floor of the lowerchassis 1100 on vibration isolators (not shown), a power supply unit1156, a power filter 1158, and fans 1160.

A different syringe pump 1152 is designated for each of the fivemagnetic separation stations 800, one is designated for the left-sideorbital mixer 552, and one is designated for the deactivation queue 750.Although syringe pumps are preferred, peristaltic pumps may be used asan alternative.

The vacuum pump 1162 services each of the magnetic separation stations800 and the deactivation queue 750. The preferred rating of the vacuumpump is 5.3-6.5 cfm at 0″ Hg and 4.2-5.2 cfm at 5″ Hg. A preferredvacuum pump is available from Thomas Industries, Inc. of Sheboygan,Wis., as Model No. 2750CGHI60. A capacitor 1172 is sold in conjunctionwith the pump 1162.

The power supply unit 1156 is preferably an ASTEC, Model No.VS1-B5-B7-03, available from ASTEC America, Inc., of Carlsbad, Calif.Power supply unit 1156 accepts 220 volts ranging from 50-60 Hz, i.e.,power from a typical 220 volt wall outlet. Power filter 1158 ispreferably a Corcom Model No. 20MV1 filter, available from Corcom, Inc.of Libertyville, Ill. Fans 1160 are preferably Whisper XLDC fansavailable from Comair Rotron, of San Ysidro, Calif. Each fan is poweredby a 24 VDC motor and has a 75 cfm output. As shown in FIG. 52, the fans1160 are preferably disposed proximate a left-side outer wall of thelower chassis 1100. The fans 1160 are preferably directed outwardly todraw air through the lower chassis from the right-side thereof to theleft-side thereof, and thus, to draw excess heat out of the lowerchassis.

Other power supply system components are housed in the back left-handside of the lower chassis 1100, including a power switch 1174,preferably an Eaton circuit breaker switch 2-pole, series JA/S,available from the Cutler-Hammer Division of Eaton Corporation ofCleveland, Ohio, and a power inlet module 1176 at which a power cord(not shown) for connecting the analyzer 50 to an external power sourceis connected. The power supply system of the analyzer 50 also includes aterminal block (not shown), for attaching thereto a plurality ofelectrical terminals, a solid state switch (not shown), which ispreferably a Crydom Series 1, Model No. D2425, available from CalSwitch, Carson City, Calif., for switching between different circuits,and an RS232 9-pin connector port for connecting the analyzer 50 to theexternal computer controller 1000.

The right-side drawer and left-side drawer bays are preferably closedbehind one or two doors (not shown) in front of the analyzer, whichis/are preferably locked by the assay manager program during operationof the analyzer. Microswitches are preferably provided to verifydoor-closed status. The far left bay is covered by a front panel. Endpanels are provided on opposite ends of the lower chassis to enclose thechassis.

Four leveler feet 1180 extend down from the four corners of the chassis1100. The leveler feet 1180 include threaded shafts with pads at thelower ends thereof. When the analyzer is in a desired location, the feet1180 can be lowered until the pads engage the floor to level andstabilize the analyzer. The feet can also be raised to permit theanalyzer to be moved on its casters.

Bulk fluids typically contained in the containers of the lower chassis1100 may include wash solution (for washing immobilized target),distilled water (for washing fixed pipette tips), diagnostic testingreagents, silicone oil (used as a floating fluid for layering over testreagents and sample), and a bleach-based reagent (used for sampledeactivation).

The right-side drawer 1104 is shown in detail in FIG. 53. The right-sidedrawer 1104 includes a box-like drawer structure with a front drawerhandle 1105. Although drawer handle 1105 is shown as a conventionalpull-type drawer handle, in the preferred embodiment of the analyzer 50,handle 1105 is a T-handle latch, such as those available from Southco,Inc. of Concordville, Pa. The drawer 1104 is mounted in the lowerchassis on slide brackets (not shown) so that the drawer 1104 can bepulled into and out of the lower chassis. A sensor (not shown) ispreferably provided for verifying that the drawer 1104 is closed. Thefront portion of the drawer includes bottle receptacles 1122 for holdingbottle 1128 (shown in FIG. 52), which is a dedicated pipette washwaste-containing bottle, and bottle 1130 (also shown in FIG. 52), whichis a dedicated waste bottle for containing waste from a magnetic wash,target-capture procedure. Bottle 1130 is preferably evacuated.

The analyzer 50 will not begin processing assays if any of the bottlesrequired in the lower chassis 1100 are missing. Bottle receptacles 1122preferably include bottle-present sensors (not shown) to verify thepresence of a bottle in each receptacle 1122. The bottle-present sensorsare preferably diffuse reflective type optical sensors available fromSUNX/Ramco Electric, Inc., of West Des Moines, Iowa, Model No. EX-14A.

Right-side drawer 1104 further includes a waste bin 1108 for holdingtherein spent MTUs and sample tips. Waste bin 1108 is an open boxstructure with a sensor mount 1112 at a top portion thereof for mountingthereon a sensor, preferably a 24 VDC Opto-diffuse reflector switch (notshown), for detecting whether the waste bin 1108 is full. Anotherdiffuse reflector type optical sensor (not shown) is positioned withinright-side drawer 1104 to verify that the waste bin 1108 is in place.Again, diffuse reflective type optical sensors available from SUNX/RamcoElectric, Inc., of West Des Moines, Iowa, Model No. EX-14A, arepreferred.

A deflector 1110 extends obliquely from a side of the waste bin 1108.Deflector 1110 is disposed directly below a chute through which spentMTUs are dropped into the waste bin 1108 and deflects the dropped MTUstoward the middle of the waste bin 1108 to avoid MTU pile-ups in acorner of the waste bin 1108. Deflector 1110 is preferably pivotallymounted so that it can pivot upwardly to a substantially verticalposition so that when a waste bag, which lines the waste bin 1108 andcovers the deflector 1110, is removed from the waste bin 1108, thedeflector 1110 will pivot upwardly with the bag as it is pulled out andtherefore will not rip the bag.

A printed circuit board (not shown) and cover 1114 can be mounted to thefront of the waste bin 1108. Sensor mounts 1116 and 1117 are alsomounted to the front of waste bin 1108. Sensors 1118 and 1119 aremounted on sensor mount 1116, and sensors 1120 and 1121 mounted onsensor mount 1117. Sensors 1118, 1119, 1120, and 1121 are preferably DCcapacitive proximity sensors. The upper sensors 1118, 1119 indicate whenthe bottles 1128 and 1130 are full, and the bottom sensors 1120, 1121indicate when the bottles are empty. Sensors 1118-1121 are preferablythose available from Stedham Electronics Corporation of Reno, Nev.,Model No. C2D45AN1-P, which were chosen because their relatively flatphysical profile requires less space within the tight confines of thelower chassis 1100 and because the Stedham sensors provide the desiredsensing distance range of 3-20 mm.

The analyzer 50 will preferably not begin performing any assays if theassay manager program detects that any of the waste fluid containers inthe right-side drawer 1104 are not initially empty.

The capacitive proximity sensors 1118-1121 and the bottle-present,waste-bin-present, and waste-bin-full optical sensors of the right-sidedrawer 1104 are connected to the printed circuit board (not shown)behind cover 1114, and the printed circuit board is connected to theembedded controller of the analyzer 50.

Because the right-side drawer 1104 cannot be pulled completely out ofthe lower chassis 1100, it is necessary to be able to pull the waste bin1108 forward so as to permit access to the waste bin for installing andremoving a waste bag liner. For this purpose, a handle 1126 is mountedto the front of the waste bin 1108 and teflon strips 1124 are disposedon the bottom floor of the right-side drawer 1104 to facilitate forwardand backward sliding of the waste bin 1108 in the drawer 1104 whenbottles 1128 and 1130 are removed.

Details of the left-side drawer 1106 are shown in FIG. 54. Left-sidedrawer 1106 includes a box-like structure with a front mounted handle1107 and is mounted within the lower chassis 1100 on slide brackets (notshown). Although handle 1107 is shown as a conventional pull-type drawerhandle, in the preferred embodiment of the analyzer 50, handle 1107 is aT-handle latch, such as those available from Southco, Inc. ofConcordville, Pa. A sensor is provided for verifying that the left-sidedrawer 1106 is closed.

Left-side drawer 1106 includes a tiplet waste bin 1134 with a mountingstructure 1135 for mounting thereon a tiplet-waste-bin-full sensor (notshown). A tiplet-waste-bin-present sensor is preferably provided in theleft-side drawer 1106 to verify that the tiplet waste bin 1134 isproperly installed. Diffuse reflective type optical sensors availablefrom SUNX/Ramco Electric, Inc., of West Des Moines, Iowa, Model No.EX-14A, are preferred for both the tiplet-waste-bin-full sensor and thetiplet-waste-bin-present sensor.

Bundling structures 1132 are provided for securing and bundling varioustubing and/or wires (not shown) within the lower chassis 1100. Thebundling structures preferably used are Energy Chain Systemsmanufactured and sold by Igus, Inc. of East Providence, R.I.

A printed circuit board 1182 is mounted behind a panel 1184 which islocated behind the tiplet waste bin 1134. A solenoid valve mountingpanel 1186 is located below the tiplet waste bin 1134.

Left-side drawer 1106 includes a forward container-holding structure forholding therein six similarly sized bottles. The container structureincludes divider walls 1153, 1155, 1157, and 1159 and container blocks1151 having a curved bottle-conforming front edge, which together definesix container-holding areas. Lower sensors 1148 and upper sensors 1150(six of each) are mounted on the divider walls 1155, 1157, and 1159. Theupper and lower sensors 1148, 1150 are preferably DC capacitiveproximity sensors (preferably sensors available from Stedham ElectronicsCorporation of Reno, Nev., Model No. C2D45AN1-P, chosen for their flatprofile and sensing range). The upper sensors 1150 indicate when thebottles held in the container structure are full, and the lower sensors1148 indicate when the bottles are empty. In the preferred arrangement,the left two bottles 1146 contain a detecting agent (“Detect I”), themiddle two bottles 1168 contain silicon oil, and the right two bottles1170 contain another detecting agent (“Detect II”).

Bottle-present sensors (not shown) are preferably provided in each ofthe container-holding areas defined by the container blocks 1151 and thedividing walls 1153, 1155, 1157, and 1159 to verify the presence ofbottles in each container-holding area. The bottle-present sensors arepreferably diffuse reflective type optical sensors available fromSUNX/Ramco Electric, Inc., of West Des Moines, Iowa, Model No. EX-14A.

A large centrally located container receptacle 1164 holds a bottle 1140(shown in FIG. 52), preferably containing deionized water. Containerreceptacles 1166 (only one is visible in FIG. 54) hold bottles 1142 and1144 (also shown in FIG. 52) preferably containing a wash solution. Adividing wall 1143 between the receptacle 1164 and 1166 has mountedthereon sensors, such as sensor 1141, for monitoring the fluid level inthe bottles 1140, 1142, and 1144. The sensors, such as sensor 1141, arepreferably DC capacitive proximity sensors (preferably sensors availablefrom Stedham Electronics Corporation of Reno, Nev., Model No.C2D45AN1-P).

Container receptacles 1164 and 1166 preferably include bottle-presentsensors (not shown) for verifying that bottles are properly positionedin their respective receptacles. The bottle-present sensors arepreferably diffuse reflective type optical sensors available fromSUNX/Ramco Electric, Inc., of West Des Moines, Iowa, Model No. EX-14A.

The analyzer 50 will not begin performing any assays if the assaymanager program determines that any of the bulk-fluid containers in theleft-side drawer 1106 are initially empty.

The capacitive proximity fluid level sensors, the various bottle-presentsensors, the tiplet-waste-bin-full sensor, and thetiplet-waste-bin-present sensors are all connected to the printedcircuit board 1182, and the printed circuit board 1182 is connected tothe embedded controller of the analyzer 50.

Four solenoid valves (not shown) are mounted below the solenoid valvemounting panel 1186. The solenoid valves connect bulk fluid bottleswhere fluids are stored in pairs of bottles, i.e., the bottles 1140,1142 containing wash solution, the two bottles 1146 containing the“Detect I” agent, the two bottles 1168 containing oil, and the twobottles 1170 containing the “Detect II” agent. The solenoid valves, inresponse to signals from the respective capacitive proximity sensors,switch bottles from which fluid is being drawing when one of the twobottles containing the same fluid is empty. In addition, the solenoidvalves may switch bottles after a prescribed number of tests areperformed. The preferred solenoid valves are teflon solenoid valvesavailable from Beco Manufacturing Co., Inc. of Laguna Hills, Calif.,Model Nos. S313W2DFRT and M223W2DFRLT. The two different model numberscorrespond to solenoid valves adapted for use with two different tubesizes. Teflon solenoid valves are preferred because they are less likelyto contaminate fluids flowing through the valves and the valves are notdamaged by corrosive fluids flowing through them.

Bottle 1136 (see FIG. 52) is a vacuum trap held in a vacuum trap bracket1137, and bottle 1138 contains a deactivating agent, such asbleach-containing reagent. Again, bottle-present sensors are preferablyprovided to verify the presence of bottles 1136 and 1138.

A hand-held bar code scanner 1190 may be provided in the lower chassis1100 for scanning information provided on scannable container labelsinto the assay manager program. Scanner 1190 is connected by a cord toprinted circuit board 1182 of the left-side drawer 1106 and ispreferably stowed on a bracket (not show) mounted on dividing wall 1143.Scanners available from Symbol Technologies, Inc., of Holtsville, N.Y.,series LS2100, are preferred.

Sample Ring and Sample Tube Trays

Samples are contained in the sample tubes 320, and the tubes 320 areloaded into the tube trays 300 outside the analyzer 50. The trays 300carrying the sample tubes 320 are placed onto the sample ring 250through the access opening provided by opening the flip-up carousel door80.

Referring to FIGS. 5 and 6, the first ring assembly, or sample ring, 250is formed of milled, unhardened aluminum and includes a raised ringstructure defining an annular trough 251 about the outer periphery ofring 250 with a plurality of raised, radially extending dividers 254extending through trough 251. Preferably, nine dividers 254 divide thetrough 251 into nine arcuate sample tube tray-receiving wells 256. Thetrough 251 and wells 256 define an annular fluid container carrierportion constructed and arranged to carry a plurality of containers aswill be described below.

Sample ring 250 is preferably rotationally supported by three120°-spaced V-groove rollers 257, 258, 260 which engage a continuousV-ridge 262 formed on the inner periphery of ring 250, as shown in FIGS.5, 6, and 6A so that the ring 250 is rotatable about a first centralaxis of rotation. The rollers are preferably made by Bishop-WisecarverCorp. of Pittsburg, Calif., Model No. W1SSX. Rollers 257 and 260 arerotationally mounted on fixed shafts, and roller 258 is mounted on abracket which pivots about a vertical axis and is spring biased so as tourge roller 258 radially outward against the inner periphery of ring250. Having two fixed rollers and one radially movable roller allows thethree rollers to accommodate an out-of-round inner periphery of the ring250.

Sample ring 250 is driven by stepper motor 264 (VEXTA stepper motorsavailable from Oriental Motor Co., Ltd. of Tokyo, Japan as Model No.PK266-01A are preferred) via continuous belt 270 (preferably availablefrom SDP/SI of New Hyde Park, N.Y., as Model No. A6R3M444080) whichextends over guide rollers 266, 268 and around the outer periphery ofring 250. A home sensor and a sector sensor (not shown), preferablyslotted optical sensors, are provided adjacent the ring 250 at arotational home position and at a position corresponding to one of thesample tube tray receiving wells 256. The ring 250 includes a home flag(not shown) located at a home position on the wheel and nineequally-spaced sector flags (not shown) corresponding to the positionsof each of the nine sample tube tray receiving wells 256. The home flagand sector flags cooperate with the home sensor and sector sensors toprovide ring position information to the assay manager program and tocontrol the ring 250 to stop at nine discrete positions corresponding toestablished coordinates for user re-load and access by pipette unit 450.Preferred sensors for the home sensor and sector sensor are Optekslotted optical sensors, Model No. OPB857, available from Optek ofCarrollton, Tex.

A sample cover is disposed over a portion of the annular fluid containercarrier portion, or trough 251, and comprises an arcuate cover plate 138fixed in an elevated position with respect to the wheel 250 on threemounting posts 136. Plate 138 has an arcuate shape generally conformingto the curve of the trough 251. A first opening 142 is formed in theplate 138, and a second opening 140 is formed in the plate 138 at agreater radial distance from the axis of rotation of ring 250 thanopening 142 and at a circumferentially-spaced position from opening 142.

Referring to FIGS. 55-57, each sample tube tray 300 comprises a testtube rack structure that is curved to conform to the curvature of thering 250. Each tray 300 comprises a central wall structure 304 withlateral end walls 303 and 305 disposed on either end of wall 304. Afloor 312 extends across the bottom of the tray 300. The principlepurposes of sample tube tray 300 are to hold sample tubes on the samplering 250 for access by the sample pipette assembly 450 and to facilitateloading and unloading of multiple sample tubes into and from theanalyzer.

A plurality of Y-shaped dividers 302 are equidistantly spaced alongopposite edges of the tray 300. Each two adjacent dividers 302 define atest-tube receiving area 330. End wall 303 includes inwardly bentflanges 316 and 318, and end wall 305 includes inwardly bent flanges 326and 328. The respective inwardly bent flanges of end walls 303 and 305along with the end-most of the dividers 302 define the end-most tubereceiving areas 332. The receiving areas 330, 332 are arcuately alignedalong two arcuate rows on opposite sides of central wall structure 304

Referring to FIG. 57, within each tube receiving area 330, 332, a leafspring element 310 is attached to central wall 304. Leaf spring element310, preferably formed of stainless spring steel, elastically deflectswhen a test tube 320 is inserted into the tube-receiving area 330 or 332and urges the tube 320 outwardly against the dividers 302. Thus, thetube 320 is secured in an upright orientation. The shape of the dividers302 and the elasticity of the leaf spring elements 310 allow the tray300 to accommodate sample tubes of various shapes and sizes, such astubes 320 and 324. Each tray 300 preferably includes nine dividers 302along each edge to form, along with end walls 303 and 305, tentube-receiving areas 330, 332 on each side of central wall structure 304for a total of twenty tube-receiving areas per tray. Indicia fordesignating tube-receiving areas 330 and 332, such as raised numerals306, may be provided on the tray, such as on central wall 304.

Each tray 300 may also include boss structures 308, shown in theillustrated embodiment to be integrally formed with the end-mostdividers 302. An upright inverted U-shaped handle (not shown) may beattached to the tray at boss structures 308 or some other suitablelocation. Upright handles can facilitate handling of the tray 300 whenloading and unloading the tray 300 through the arcuate carousel door 80,but are not necessarily preferred.

A gap is provided between adjacent dividers 302 so that bar-code labels334, or other readable or scannable information, on the tubes 320 isaccessible when the tube is placed in the tray 300. When a tray 300carried on wheel 250 passes beneath the plate 138 of the sample cover,one tube 320 in a curved row at a radially-inward position with respectto wall structure 304 will be aligned with first opening 142 and anothertube 320 in a curved row at a radially-outward position with respect towall 304 will be aligned with second opening 140. The ring 250 isindexed to sequentially move each tube 320 beneath the openings 140, 142to permit access to the tubes.

Referring again to FIG. 5, bar code scanners 272 and 274 are disposedadjacent the ring 250. Opticon, Inc. scanners, Model No.LHA2126RR1S-032, available from Opticon, Inc. of Orangeburg, N.Y., arepreferred. Scanner 272 is located outside ring 250, and scanner 274 isdisposed inside ring 250. Scanners 272 and 274 are positioned to scanbar code data labels on each sample tube 320 carried in the sample tubetray 300 as the ring 250 rotates a tray 300 of sample tubes 320 past thescanners 272, 274. In addition, the scanners 272, 274 scan the bar codelabel 337 (see FIG. 55) on the outer portion of bent flanges 316 and 318of end wall 303 of each tray 300 as the tray 300 is brought into thesample preparation area. Various information, such as sample and assayidentification, can be placed on the tubes and/or each tray 300, andthis information can be scanned by the scanners 272, 274 and stored inthe central processing computer. If no sample tube is present, the tray300 presents a special code 335 (see FIG. 55) to be read by the scanners272, 274.

A preferred sample tube holder is disclosed by Knight et al., “SampleTube Holder,” U.S. Provisional Application No. 60/672,609, which enjoyscommon ownership herewith. Knight discloses sample tube holders having aplurality of sample tube compartments with aligned sets of fingersprings for holding sample tubes in fixed, vertical orientations. Forapplications in which the sample tubes are capped with penetrableclosures, the sample tube holders include a retainer for maintainingsample tubes within the sample tube compartments during samplingprocedures. See, e.g., Kacian et al., “Penetrable Cap,” U.S. Pat. No.6,893,612 (discloses a sample tube closed with a cap having a frangibleseal and a filter for limiting the dissemination of a contaminatingaerosol or bubbles).

Pipette Tip Wheel

As shown primarily in FIGS. 5 and 6, a second ring assembly of thepreferred embodiment is a pipette tip wheel 350 and comprises a circularring 352 at a bottom portion thereof, a top panel 374 defining acircular inner periphery and five circumferentially-spaced,radially-protruding sections 370, and a plurality of generallyrectangular risers 354 separating the top panel 374 from the ring 352and preferably held in place by mechanical fasteners 356 extendingthrough the top panel 374 and ring 352 into the risers 354. Fiverectangular openings 358 are formed in the top panel 374 proximate eachof the sections 370, and a rectangular box 376 is disposed beneath panel374, one at each opening 358. Top panel 374, ring 352, and risers 354are preferably made from machined aluminum, and boxes 376 are preferablyformed from stainless steel sheet stock.

The openings 358 and associated boxes 376 are constructed and arrangedto receive trays 372 holding a plurality of disposable pipette tips. Thepipette tip trays 372 are preferably those manufactured and sold byTECAN (TECAN U.S. Inc., Research Triangle Park, N.C.) under the tradename “Disposable Tips for GENESIS Series”. Each tip has a 1000 μlcapacity and is conductive. Each tray holds ninety-six elongateddisposable tips.

Lateral slots 378 and longitudinal slots 380 are formed in the top panel374 along the lateral and longitudinal edges, respectively, of eachopening 358. The slots 378, 380 receive downwardly-extending flanges(not shown) disposed along the lateral and longitudinal edges of thetrays 372. The slots 378, 380 and associated flanges of the trays 372serve to properly register the trays 372 with respect to openings 358and to hold the trays 372 in place on the panel 374.

Pipette tip wheel 350 is preferably rotationally supported by three120°-spaced V-groove rollers 357, 360, 361 which engage a continuousV-ridge 362 formed on the inner periphery of ring 352, as shown in FIGS.5, 6, and 6A, so that the pipette tip wheel 350 is rotatable about asecond central axis of rotation that is generally parallel to the firstaxis of rotation of the sample ring 250. The rollers are preferably madeby Bishop-Wisecarver Corp. of Pittsburg, Calif., Model No. W1SSX.Rollers 357 and 360 are rotationally mounted on fixed shafts, and roller361 is mounted on a bracket which pivots about a vertical axis and isspring biased so as to urge roller 361 radially outwardly against theinner periphery of ring 352. Having two fixed rollers and one radiallymovable roller allows the three rollers to accommodate an out-of-roundinner periphery of ring 352. In addition, the wheel 350 can be easilyinstalled and removed by merely pushing pivoting roller 361 radiallyinwardly to allow the ring 352 to move laterally to disengage continuousV-ridge 362 from the fixed V-groove rollers 357, 360.

Pipette tip wheel 350 is driven by a motor 364 having a shaft-mountedspur gear which meshes with mating gear teeth formed on an outerperimeter of ring 352. Motor 364 is preferably a VEXTA gear head steppermotor, Model No. PK243-A1-SG7.2, having a 7.2:1 gear reduction andavailable from Oriental Motor Co., Ltd. of Tokyo, Japan. A gear headstepper motor with a 7.2:1 gear reduction is preferred because itprovides smooth motion of the pipette tip wheel 350, where the spur gearof the motor 364 is directly engaged with the ring 352.

A home sensor and a sector sensor (not shown), preferably slottedoptical sensors, are provided adjacent the pipette tip wheel 350 at arotational home position and at a position of one of the boxes 376. Thepipette tip wheel 350 includes a home flag (not shown) located at a homeposition on the wheel and five equally-spaced sector flags (not shown)corresponding to the positions of each of the five boxes 376. The homeflag and sector flags cooperate with the home sensor and sector sensorsto provide wheel position information to the assay manager program andto control the pipette tip wheel 350 to stop at five discrete positionscorresponding to established coordinates for user re-load and access bypipette unit 450. Preferred sensors for the home sensor and sectorsensor are Optek Technology, Inc. slotted optical sensors, Model No.OPB980, available from Optek Technology, Inc. of Carrollton, Tex.

Multi-Axis Mixer

Referring to FIGS. 7-12, the multi-axis mixer 400 includes a rotatingturntable structure 414 (see FIG. 10) rotatably mounted on a centershaft 428 supported in center bearings 430 to a fixed base 402 mountedto the jig plate 130 by means of mechanical fasteners (not shown)extending through apertures 419 formed about the outer periphery of thefixed base 402. A cover member 404 is attached to and rotates withturntable 414.

Turntable 414 is preferably in the form of a right angle crosscomprising three 90-spaced rectangular arms 444 of equal lengthextending radially outwardly from the center of the turntable 414 and afourth arm 445 having an extension 417 making arm 445 slightly longerthan arms 444. As shown in FIGS. 10-12, the center portion of turntable414 is connected to center shaft 428 by a screw 429.

Four container holders 406 are disposed on the ends of the arms 444 and445 of turntable frame 414. Each container holder 406 is attached to oneof four vertical shafts 423, which are rotatably supported in containerholder bearings 415. Container holder bearings 415 are pressed into thearms 444, 445 of the turntable 414 and are disposed at equal radialdistances from shaft 428.

The cover member 404 includes four circular openings withupwardly-turned peripheral flanges 401 through which shafts 423 extend.Upward flanges 401 can advantageously prevent spilled liquids fromflowing into the openings.

The container holders 406 comprise generally cylindrical members havingan open bottom and an open top for receiving and holding a container440, preferably a plastic bottle, of target capture reagent.

The target capture reagent used with the preferred assay includesmagnetically responsive particles with immobilized polynucleotides,polynucleotide capture probes, and reagents sufficient to lyse cellscontaining the targeted nucleic acids. After cell lysis, targetednucleic acids are available for hybridization under a first set ofpredetermined hybridization conditions with one or more capture probes,with each capture probe having a nucleotide base sequence region whichis capable of hybridizing to a nucleotide base sequence region containedin at least one of the targeted nucleic acids. Under a second set ofpredetermined hybridization conditions, a homopolymer tail (e.g.,oligo(dT)) of the immobilized polynucleotides is capable of hybridizingwith a complementary homopolymer tail (e.g., oligo(dA)) contained in thecapture probe, thereby immobilizing targeted nucleic acids. Varioustarget-capture methods and lysing procedures are well known in the artand are readily adapted for use with the analyzer 50 of the presentinvention. This preferred two-step capture method of capturing andimmobilizing a target nucleic acid on a magnetically responsive particleis disclosed by Weisburg et al. in U.S. Pat. No. 6,534,273.

A container retainer spring 408 spans a lateral slot formed in the wallof each container holder 406 and helps to hold the container 440 withinthe container holder 406 by urging the container 440 toward a portion ofthe inner peripheral wall of the holder 406 opposite the spring 408.

Each container holder 406 is secured to an associated vertical shaft 423by a shaft block structure 432. Shaft block structure 432 includescurved end portions which conform to the inside of the cylindricalcontainer holder 406, and the container holder 406 is secured to theblock 432 by fasteners 434. A generally circular aperture 449 receivesthe shaft 423. A slot 438 extends from aperture 449 to an end of theblock 432 which does not extend all the way to the inside of thecontainer holder 406, and a second slot 436 extends from an edge of theblock 432 generally perpendicularly to slot 438 so as to define acantilevered arm 435. A machine screw 437 extends through a through-hole441 formed laterally through block 432 and into a threaded hole 447formed laterally through arm 435. As screw 437 is tightened, arm 435deflects, thus tightening aperture 449 around shaft 423.

The shaft block structure 432, the shaft 423, and the container holderbearings 415 associated with each container holder 406 define apreferred container holder mounting structure associated with eachcontainer holder 406 that is constructed and arranged to mount thecontainer holder 406 to the turntable 414 and permit the containerholder 406 to rotate about an axis of rotation 412 of the shaft 423.

Container holder planetary gears 422 are attached to the opposite endsof shafts 423. The planetary gears 422 operatively engage a stationarysun gear 416. A drive pulley 418 is attached to center shaft 428 and iscoupled to a drive motor 420 by a drive belt (not shown). Drive motor420 is preferably mounted so as to extend through an opening (not shown)in the jig plate 130 below the base 402. Drive motor 420 is preferably astepper motor, and most preferably a VEXTA stepper motor, Model No.PK264-01A, available from Oriental Motor Co., Ltd. of Tokyo, Japan. Thedrive motor 420, via the drive belt and drive pulley 418, rotates thecenter shaft 428 and the turntable 414 attached thereto. As theturntable frame 414 rotates about the center line of center shaft 428,the planetary gears 422 engaged with sun gear 416 cause the shafts 423and container holders 406 attached thereto to rotate at the ends of thearms 444 of the turntable frame 414. Each container holder 406 ispreferably mounted such that the axis of rotation 410 thereof is offsetfrom the axis of rotation 412 of the associated shaft 423. Thus, eachcontainer holder 406 rotates eccentrically about axis 412 of theassociated shaft 423. Accordingly, the planetary gears 422 and the sungear 416 constitute rotational motion coupling elements constructed andarranged to cause the container holders 406 to rotate about therespective axes of rotation of the shafts 423 as the turntable 414rotates about the axis of rotation of the shaft 428.

A bar code scanner device 405 is preferably mounted on a bracket 403 andreads bar code information of the containers 440 through a scanner slot407 formed in each container holder 406. The preferred scanner is aModel No. NFT1125/002RL scanner, available from Opticon, Inc. ofOrangeburg, N.Y.

The multi-axis mixer 400 usually rotates during operation of theanalyzer 50 to agitate the fluid contents of the containers 440 tothereby keep the target capture reagent in suspension, stopping onlybriefly to permit pipette unit 456 to withdraw an amount of mixture fromone of the containers. Pipette unit 456 draws mixture from a bottle atthe same location each time. Therefore, it is desirable to monitor thepositions of the bottles so that the bottle from which mixture iswithdrawn each time can be specified.

Four optical slotted sensors 426, each comprising an optical emitter anddetector, are stationed around the periphery of fixed base 402, spacedat 90° intervals. Optical sensors available from Optek Technology, Inc.of Carrollton, Tex., Model No. OPB490P11, are preferred. A sensor tab424 extends down from extension 417 at the end of arm 445 of theturntable 414. When sensor tab 424 passes through a sensor 426, thecommunication between the emitter and detector is broken thus giving a“container present” signal. The tab 424 is only provided at onelocation, e.g., the first container location. By knowing the position ofthe first container, the positions of the remaining containers, whichare fixed relative to the first container, are also known.

Power and control signals are provided to the multi-axis mixer 400 via apower and data connector. While the multi-axis mixer 400 provides mixingby rotation and eccentric revolution, other mixing techniques, such asvibration, inversion, etc. may be used.

Sample Preparation Procedure

To begin sample preparation, the pipette unit 456 moves to transfertarget capture reagent, preferably mag-oligo reagent, from a container440 carried on the multi-axis mixer 400 into each of the reaction tubes162 of the MTU 160. The target capture reagent includes a supportmaterial able to bind to and immobilize a target analyte. The supportmaterial preferably comprises magnetically responsive particles. At thebeginning of the sample preparation procedure, the pipette unit 456 ofthe right-side pipette assembly 450 moves laterally and longitudinallyto a position in which the probe 457 is operatively positioned over apipette tip in one of the trays 372.

The tip trays 372 are carried on the pipette tip wheel 350 so as to beprecisely positioned to achieve proper registration between the pipettetips and the tubular probe 457 of the pipette unit 456. The pipette unit456 moves down to insert the free end of the tubular probe 457 into theopen end of a pipette tip and frictionally engage the pipette tip. TheCavro processors preferably used for pipette unit 456 includes a collar(not shown), which is unique to Cavro processors. This collar is movedslightly upwardly when a pipette tip is frictionally engaged onto theend of the tubular probe 457, and the displaced collar trips anelectrical switch on the pipette unit 456 to verify that a pipette tipis present. If tip pick-up is not successful (e.g., due to missing tipsin the trays 372 or a misalignment), a missing tip signal is generatedand the pipette unit 456 can move to re-try tip engagement at adifferent tip location.

The assay manager program causes the multi-axis mixer 400 to brieflystop rotating so that the pipette unit 456 can be moved to a positionwith the tubular probe 457 and attached pipette tip of the pipette unit456 aligned over one of the stationary containers 440. The pipette unit456 lowers the pipette tip attached to the tubular probe 457 into thecontainer 440 and draws a desired amount of target capture reagent intothe pipette tip. The pipette unit 456 then moves the probe 457 out ofthe container 440, the multi-axis mixer 400 resumes rotating, and thepipette unit 456 moves to a position above opening 252 and the sampletransfer station 255. Next, the pipette unit 456 descends, moving thepipette tip and the tubular probe 457 through the opening 252, anddispenses a required amount of target capture (typically 100-500 μl)into one or more of the reaction tubes 162 of the MTU 160. It ispreferred that the target capture reagent is drawn only into the pipettetip and not into the probe 457 itself. Furthermore, it is preferred thatthe pipette tip be of sufficient volumetric capacity to hold enoughreagent for all five reaction tubes 162 of the MTU 160.

After target capture reagent transfer, the pipette unit 456 then movesto a “tip discard” position above tip disposal tube 342, where thedisposable pipette tip is pushed or ejected off of the end of thetubular probe 457 of the pipette unit 456, and falls through tube 342toward a solid waste container. An optical sensor (not shown) isdisposed adjacent to tube 342, and before tip discard, the samplepipette assembly 450 moves the pipette unit 456 into a sensing positionof the sensor. The sensor detects whether a tip is engaged with the endof the tubular probe 457 to verify that the tip is still held on thetubular probe 457 of the pipette unit 456, thereby confirming that thetip was on the tubular probe 457 throughout sample preparation. Apreferred sensor is a wide-gap slotted optic sensor, Model No. OPB900W,available from Optek Technology, Inc. of Carrollton, Tex.

Preferably, the pipette tip is ejected by the collar (not shown) on thetubular probe 457 of pipette unit 456. The collar engages a hard stopwhen the tubular probe 457 is raised, so that as the probe 457 continuesto ascend, the collar remains fixed and engages an upper end of thepipette tip, thereby forcing it off the tubular probe 457.

After pipetting the target capture and discarding the pipette tip, theprobe 457 of the pipette unit 456 can be washed by running distilledwater through the tubular probe 457 at the tip wash station basin 346.The tip wash water is collected and drains down into a liquid wastecontainer.

Following the reagent dispensing procedure, the pipette unit 456 on theright pipette assembly 450 moves laterally and longitudinally to aposition in which the tubular probe 457 of the pipette unit 456 iscentered over a new pipette tip on one of the tip trays 372. Aftersuccessful tip engagement, the pipette unit 456 moves back over thesample ring 250, adjacent to the sample preparation opening 252 andwithdraws a test sample (about 25-900 μl) from a sample tube 320 that isaligned with one of the openings 140, 142 of the cover plate 138. Notethat both openings 140, 142 include upwardly extending peripheralflanges to prevent any fluids spilled onto the plate 138 from runninginto the openings 140, 142. The pipette unit 456 then moves over the MTU160 in the sample transfer station 255, moves down through opening 252,and dispenses test sample into one of the reaction tubes 162 of the MTU160 containing target capture reagent. Pipette unit 456 then moves tothe “tip discard” position above the tip disposal tube 342, and thedisposable pipette tip is ejected into the tube 342. Pipette unit 456then picks up a new disposable pipette tip from the pipette tip wheel350, the sample ring 250 indexes so that a new sample tube is accessibleby the pipette unit 456, unit 456 moves to and draws sample fluid fromthe sample tube into the disposable pipette tip, the pipette unit 456then moves to a position above the sample transfer station 255, anddispenses sample fluid into a different reaction tube 162 containingtarget capture reagent. This process is preferably repeated until allfive reaction tubes 162 contain a combination of fluid sample sample andtarget capture reagent.

Alternatively, depending on the assay protocol or protocols to be run bythe analyzer 50, the pipette unit 456 may dispense the same test samplematerial into two or more of the reaction tubes 162 and the analyzer canperform the same or different assays on each of those aliquots.

As described above with respect to pipette units 480, 482, pipette unit456 also includes capacitive level sensing capability. The pipette tipsused on the end of the tubular probe 457 are preferably made from aconductive material, so that capacitive level sensing can be performedwith the pipette unit 456, even when a tip is carried on the end of thetubular probe 457. After the pipette unit has completed a test sampledispensing procedure, the pipette unit 456 moves the tubular probe 457back down into the reaction tube 162 until the top of the fluid level isdetected by the change in capacitance. The vertical position of thetubular probe 457 is noted to determine whether the proper amount offluid material is contained in the reaction tube 162. Lack of sufficientmaterial in a reaction tube 162 can be caused by clotting in the testsample, which can clot the tip at the end of the tubular probe 457 andprevent proper aspiration of test sample material into the tip and/orcan prevent proper dispensing of test sample from the tip.

After sample transfer, the pipette tip is discarded into the tipdisposal tube 342 as described above. Again, the tubular probe 457 ofthe pipette of unit can be washed with distilled water if desired, butwashing of the probe is typically not necessary because, in thepreferred method of operation, sample material only comes into contactwith the disposable pipette tip.

The assay manager program includes pipette unit control logic whichcontrols movements of the pipette units 456, 480, 482, and preferablycauses pipette unit 456 to move in such a manner that it never passesover a sample tube 320 on the sample ring 250, except when the pipetteunit 456 positions the tubular probe 457 over a sample tube 320 towithdraw a test sample or when the sample tube 320 is below the plate138 of the sample cover. In this way, inadvertent fluid drips from thetubular probe 457 of the pipette unit 450 into another sample tube,which might result in cross-contamination, are avoided.

Following sample preparation, the MTU 160 is moved by the right-sidetransport mechanism 500 from the sample transfer station to the rightorbital mixer 550 in which the sample/reagent mixtures are mixed. Thestructure and operation of the orbital mixers 550, 552 will be describedin further detail below.

After the MTU 160 is withdrawn from the sample transfer station by theright-side transport mechanism 500, the reaction receptacle shuttleassembly within the input queue 150 advances the next MTU into aposition to be retrieved by the right-side transport mechanism 500 whichmoves the next MTU to the sample transfer station. Sample preparationprocedures are then repeated for this next MTU.

Transport Mechanisms

The right-side and left-side transport mechanisms 500, 502 will now bedescribed in detail. Referring to FIGS. 13-16, the right-side transportmechanism 500 (as well as the left-side transport mechanism 502) has amanipulating hook member that, in the illustrated embodiment, includesan extendible distributor hook 506 extending from a hook mountingstructure 508 that is radially and slidably displaceable in a slot 510on a plate 512. A housing 504 on top of the plate 512 has an opening 505configured to receive the upper portion of an MTU 160. A stepper motor514 mounted on the plate 512 turns a threaded shaft 516, which, incooperation with a lead screw mechanism, moves the distributor hook 506from the extended position shown in FIGS. 13 and 15, to the retractedposition shown in FIG. 14, the motor 514 and threaded shaft 516constituting elements of a preferred hook member drive assembly. Steppermotor 514 is preferably a modified HIS, series 46000. HIS stepper motorsare available from Haydon Switch and Instrument, Inc. of Waterbury,Conn. The HIS motor is modified by machining the threads off one end ofthe threaded shaft 516, so that the shaft 516 can receive the hookmounting structure 508.

The housing 504, motor 514, and the plate 512 are preferably covered bya conforming shroud 507.

As shown in FIG. 16, a stepper motor 518 turns a pulley 520 via a belt519. (VEXTA stepper motors, Model No. PK264-01A, available from OrientalMotor Co., Ltd. of Tokyo, Japan, and SDP timing belts, Model No.A6R51M200060, available from SDP/SI of New Hyde Park, N.Y., arepreferred). Pulley 520 is preferably a custom-made pulley with onehundred sixty-two (162) axial grooves disposed around its perimeter. Amain shaft 522 fixedly attached to the plate 512, by means of auniquely-shaped mounting block 523, extends down through a base 524 andis fixed to the pulley 520. Base 524 is mounted to the datum plate 82 bymeans of mechanical fasteners extending through apertures 525 formedabout the outer periphery of the base 524. A flex circuit 526 providespower and control signals to the hook mounting structure 508 and motor514, while allowing the plate 512 (and the components carried on theplate) to pivot sufficiently so as to rotate as much as 340° withrespect to the base 524. The transport mechanism 500, 502, assemblypreferably includes hard stops (not shown) at either end of the unit'srotational path of travel.

An arm position encoder 531 is preferably mounted on an end of the mainshaft 522. The arm position encoder is preferably an absolute encoder.A2 series encoders from U.S. Digital in Seattle, Wash., Model No.A2-S-K-315-H, are preferred.

The assay manager program provides control signals to the motors 518 and514, and to the hook mounting structure 508, to command the distributorhook 506 to engage the MTU manipulating structure 166 on MTU 160. Withthe hook 506 engaged, the motor 514 can be energized to rotate the shaft516 and thereby withdraw the hook 506, and the MTU 160, back into thehousing 504. The MTU 160 is securely held by the transport mechanism500, 502 via the sliding engagement of the connecting rib structure 164of the MTU 160 with opposed edges 511 of plate 512 adjacent slot 510.The plate 512 thereby constitutes an element of a preferred receptaclecarrier assembly that is constructed and arranged to be rotatable aboutan axis of rotation (e.g., the axis of shaft 522) and to receive andcarry a reaction receptacle (e.g., MTU 160). The motor 518 can rotatethe pulley 520 and shaft 522 via the belt 519 to thereby rotate theplate 512 and housing 504 with respect to the base 524. Rotation of thehousing 504 thus changes the orientation of the engaged MTU, therebybringing that MTU into alignment with a different station on theprocessing deck.

Sensors 528, 532 are provided in opposite sides of the housing 504 toindicate the position of the distributor hook 506 within the housing504. Sensor 528 is an end-of-travel sensor, and sensor 532 is a homesensor. Sensors 528, 532 are preferably optical slotted sensorsavailable from Optek Technology, Inc. of Carrollton, Tex., Model No.OPB980T11. For the home sensor 532, the sensor beam is broken by a homeflag 536 extending from the hook mounting structure 508 when the hook506 is in its fully retracted position. The beam of the end-of-travelsensor 528 is broken by an end-of-travel flag 534 extending from theopposite side of the hook mounting structure 508 when the hook 506 isfully extended.

An MTU-present sensor 530 mounted in the side of the housing 504 sensesthe presence of an MTU 160 in the housing 504. Sensor 530 is preferablya SUNX, infra-red sensor, available from SUNX/Ramco Electric, Inc., ofWest Des Moines, Iowa.

Temperature Ramping Stations

One or more temperature ramping stations 700 are preferably disposedbelow the jig plate 130 and sample ring 250 (no temperature rampingstations located below the sample ring 250 are shown in the figures).After mixing the contents of the MTU 160 within the orbital mixer 550,the right-side transport mechanism 500 may move the MTU 160 from theright orbital mixer 550 to a temperature ramping station 700, dependingon the assay protocol.

The purpose of each ramping station 700 is to adjust the temperature ofan MTU 160 and its contents up or down as desired. The temperature ofthe MTU and its contents may be adjusted to approximate an incubatortemperature before inserting the MTU into the incubator to avoid largetemperature fluctuations within the incubator.

As shown in FIGS. 17-18, a temperature ramping station 700 includes ahousing 702 in which an MTU 160 can be inserted. The housing 702includes mounting flanges 712, 714 for mounting the ramping station 700to the datum plate 82. A thermoelectric module 704 (also known as aPeltier device) in thermal contact with a heat sink structure 706 isattached to the housing 702, preferably at the bottom 710. Preferredthermoelectric modules are those available from Melcor, Inc. of Trenton,N.J., Model No. CP1.4-127-06L. Although one thermoelectric module 704 isshown in FIG. 17, the ramping station 700 preferably includes two suchthermoelectric modules. Alternatively, the outer surface of the housing702 could be covered with a mylar film resistive heating foil material(not shown) for heating the ramping station. Suitable mylar film heatingfoils are etched foils available from Minco Products, Inc. ofMinneapolis, Minn. and from Heatron, Inc. of Leavenworth, Kans. Forramp-up stations (i.e., heaters), resistive heating elements arepreferably used, and for ramp-down stations (i.e., chillers),thermoelectric modules 704 are preferably used. The housing 702 ispreferably covered with a thermal insulating jacket structure (notshown).

The heat sink structure used in conjunction with the thermoelectricmodule 704 preferably comprises an aluminum block with heat dissipatingfins 708 extending therefrom.

Two thermal sensors (not shown) (preferably thermistors rated 10 KOhm at25° C.) are preferably provided at a location on or within the housing702 to monitor the temperature. YSI 44036 series thermistors availablefrom YSI, Inc. of Yellow Springs, Ohio are preferred. YSI thermistorsare preferred because of their high accuracy and the ±0.1° C.interchangeability provided by YSI thermistors from one thermistor toanother. One of the thermal sensors is for primary temperature control,that is, it sends signals to the embedded controller for controllingtemperature within the ramping station, and the other thermal sensor isfor monitoring ramping station temperature as a back-up check of theprimary temperature control thermal sensor. The embedded controllermonitors the thermal sensors and controls the heating foils or thethermoelectric module of the ramping station to maintain a generallyuniform, desired temperature within the ramping station 700.

An MTU 160 can be inserted into the housing, supported on the MTUsupport flanges 718 which engage the connecting rib structure 164 of theMTU 160. A cut-out 720 is formed in a front edge of a side panel of thehousing 702. The cut-out 720 permits a distributor hook 506 of atransport mechanism 500 or 502 to engage or disengage the MTUmanipulating structure 166 of an MTU 160 inserted all the way into atemperature ramping station 700 by lateral movement with respectthereto.

Rotary Incubators

Continuing with the general description of the assay procedure,following sufficient temperature ramp-up in a ramping station 700, theright-side transport mechanism 500 retrieves the MTU from the rampingstation 700 and places the MTU 160 into the TC incubator 600. In apreferred mode of operation of the analyzer 50, the TC incubator 600incubates the contents of the MTU 160 at about 60° C. For certain tests,it is important that the annealing incubation temperature not vary morethan ±0.5° C. and that amplification incubation (described below)temperature not vary more than ±0.1° C. Consequently, the incubators aredesigned to provide a consistent uniform temperature.

The details of the structure and operation of the two embodiments of therotary incubators 600, 602, 604 and 606 will now be described. Referringto FIGS. 19-23C, each of the incubators has housing with a generallycylindrical portion 610, suitably mounted to the datum plate 82, withinan insulating jacket 612 and an insulated cover 611.

The cylindrical portion 610 is preferably constructed of nickel-platedcast aluminum and the metal portion of the cover 611 is preferablymachined aluminum. The cylindrical portion 610 is preferably mounted tothe datum plate 82 atop three or more resin “feet” 609. The feet 609 arepreferably formed of Ultem®-1000 supplied by General Electric Plastics.The material is a poor thermal conductor, and therefore the feet 609function to thermally isolate the incubator from the datum plate. Theinsulation 612 and the insulation for the cover 611 are preferablycomprised of ½ inch thick polyethylene supplied by the Boyd Corporationof Pleasantown, Calif.

Receptacle access openings 614, 616 are formed in the cylindricalportion 610, and cooperaing receptacle access openings 618, 620 areformed in the jacket 612. For incubators 600 and 602, one of the accessopenings is positioned to be accessible by the right-side transportmechanism 500 and the other access opening is positioned to beaccessible by the left-side transport mechanism 502. Incubators 604 and606 need to be accessible only by the left-side transport mechanism 502and therefore only have a single receptacle access opening.

Closure mechanisms comprising revolving doors 622, 624 are rotatablypositioned within the openings 614 and 616. Each revolving door 622, 624has a MTU slot 626 extending through a solid cylindrical body. The MTUslot 626 is configured to closely match the profile of the MTU 160,having a wider upper portion compared to the lower portion. A doorroller 628, 630 is attached on top of each of the doors 622, 624,respectively. The revolving doors 622, 624 are actuated by solenoids(not shown) which are controlled by commands from the assay managerprogram to open and close the doors 622, 624 at the proper times. A door622 or 624 is opened by turning the door 622, 624 so that the MTU slot626 thereof is aligned with the respective receptacle access opening614, 616 and is closed by turning the door 622, 624 so that the MTU slot626 thereof extends transversely to the respective access opening 614,616. The cylindrical portion 610, cover 611, doors 622, 624, and a floorpanel (not shown) constitute an enclosure which defines the incubationchamber.

The doors 622, 624 are opened to permit insertion or retrieval of an MTUinto or from an incubator and are closed at all other times to minimizeheat loss from the incubator through the access openings 614, 616.

A centrally positioned radial fan 632 is driven by an internal fan motor(not shown). A Papst, Model No. RER 100-25/14 centrifugal fan, availablefrom ebm/Papst of Farmington, Conn., having a 24 VDC motor and rated at32 cfm is preferred because its shape is well-suited to applicationwithin the incubator.

Referring now to FIG. 22, an MTU carousel assembly 671 is a preferredreceptacle carrier which carries a plurality of radially oriented,circumferentially-arranged MTUs 160 within the incubator. The MTUcarousel assembly 671 is carried by a top plate 642, which is supportedby the cylindrical portion 610 of the housing, and is preferablyactuated by a rotation motor 640, preferably a stepper motor, supportedat a peripheral edge of on the top plate 642. Rotation motor 640 ispreferably a VEXTA stepper motor, Model No. PK246-01A, available fromOriental Motor Co., Ltd. of Tokyo, Japan.

The MTU carousel 671 includes a hub 646 disposed below the top plate 642and coupled, via a shaft 649 extending through the top plate 642, to apulley 644. Pulley 644 is preferably a custom-made pulley with onehundred sixty-two (162) axial grooves disposed around its perimeter andis coupled to motor 640 through a belt 643, so that motor 640 can rotatethe hub 646. Belt 643 is preferably a GT® series timing belt availablefrom SDP/SI of New Hyde Park, N.Y. A 9:1 ratio is preferably providedbetween the pulley 644 and the motor 640. The hub 646 has a plurality ofequally spaced-apart internal air flow slots 645 optionally separated byradially-oriented, circumferentially arranged divider walls 647. In theillustration, only three divider walls 647 are shown, although it willbe understood that divider walls may be provided about the entirecircumference of the hub 646. In the preferred embodiment, divider walls647 are omitted. A support disk 670 is attached to hub 646 and disposedbelow top plate 642 in generally parallel relation therewith. Aplurality of radially extending, circumferentially-arranged MTU holdingmembers 672 are attached to the bottom of the support disk 670 (onlythree MTU holding members 672 are shown for clarity). The MTU holdingmembers 672 have support ridges 674 extending along opposite sidesthereof. Radially oriented MTUs are carried on the MTU carousel assembly671 within stations 676 defined by circumferentially adjacent MTUholding members 672, with the support ridges 674 supporting theconnecting rib structures 164 of each MTU 160 carried by the MTUcarousel assembly 671.

The MTU carousel assembly rotates on a carousel drive shaft to which thedrive pulley (644 in the illustrated embodiment) is attached. A carouselposition encoder is preferably mounted on an exterior end of thecarousel drive shaft. The carousel position encoder preferably comprisesa slotted wheel and an optical slot switch combination (not shown). Theslotted wheel can be coupled to the carousel assembly 671 to rotatetherewith, and the optical slot switch can be fixed to the cylindricalportion 610 of the housing or top plate 642 so as to be stationary. Theslotted wheel/slot switch combination can be employed to indicate arotational position of the carousel assembly 671 and can indicate a“home” position (e.g., a position in which an MTU station 676 designatedthe #1 station is in front of the access opening 614). A2 seriesencoders from U.S. Digital in Seattle, Wash., Model No. A2-S-K-315-H,are preferred.

A heat source is provided in thermal communication with the incubatorchamber defined within the incubator housing comprising the cylindricalportion 610 and cover 611. In the preferred embodiment, Mylarfilm-encased electrically-resistive heating foils 660 surround thehousing 610 and may be attached to the cover 611 as well. Preferredmylar film heating foils are etched foils available from Minco Products,Inc. of Minneapolis, Minn. and Heatron, Inc. of Leavenworth, Kans.Alternative heat sources may include internally mounted resistiveheating elements, thermal-electric heating chips (Peltiers), or a remoteheat-generating mechanism thermally connected to the housing by aconduit or the like.

As shown in FIGS. 19 and 22, a pipette slot 662 extends through theincubator cover 611, radially-aligned pipette holes 663 extend throughthe top plate 642, and pipettes slots 664 are formed in the support disk670 over each MTU station 676, to allow pipetting of reagents into MTUsdisposed within the incubators. In the preferred embodiment of theanalyzer 50 for the preferred mode of operation, only two of theincubators, the AMP incubator 604 and the hybridization protection assayincubator HYB incubator, include the pipette holes 663 and pipette slots662 and 664, because, in the preferred mode of operation, it is only inthese two incubators where fluids are dispensed into MTUs 160 while theyare in the incubator.

Two temperature sensors 666, preferably thermistors (10 KOhm at 25° C.),are positioned in the top plate 642. YSI 44036 series thermistorsavailable from YSI, Inc. of Yellow Springs, Ohio are preferred. YSIthermistors are preferred because of their high accuracy and the ±0.1°C. interchangeability provided by YSI thermistors from one thermistor toanother. One of the sensors 666 is for primary temperature control, thatis, it sends singles to the embedded controller for controllingtemperature within the incubator, and the other sensor is for monitoringtemperature of the incubator as a back-up check of the primarytemperature control sensor. The embedded controller monitors the sensors666 and controls the heating foils 660 and fan 632 to maintain auniform, desired temperature within the incubator housing 610.

As a transport mechanism 500, 502 prepares to load an MTU 160 into anincubator 600, 602, 604, or 606, the motor 640 turns the hub 646 tobring an empty MTU station 676 into alignment with the receptacle accessopening 614 (or 616). As this occurs, the door-actuating solenoidcorrespondingly turns the revolving door 622 (or 624) one-quarter turnto align the MTU slot 626 of the door with the MTU station 676. Theaccess opening 614 is thus exposed to allow placement or removal of anMTU 160. The transport mechanism 500 or 502 then advances thedistributor hook 506 from the retracted position to the extendedposition, pushing the MTU 160 out of the housing 504, through the accessopening 614, and into an MTU station 676 in the incubator. After thedistributor hook 506 is withdrawn, the motor 640 turns the hub 646,shifting the previously inserted MTU 160 away from the access opening614, and the revolving door 622 closes once again. This sequence isrepeated for subsequent MTUs inserted into the rotary incubator.Incubation of each loaded MTU continues as that MTU advances around theincubator (counter-clockwise) towards the exit slot 618.

An MTU sensor (preferably an infrared optical reflective sensor) in eachof the MTU stations 676 detects the presence of an MTU 160 within thestation. Optek Technology, Inc. sensors, Model No. OPB770T, availablefrom Optek Technology, Inc. of Carrollton, Tex. are preferred because ofthe ability of these sensors to withstand the high temperatureenvironment of the incubators and because of the ability of thesesensors to read bar code data fixed to the label-receiving surfaces 175of the label-receiving structures 174 of the MTUs 160. In addition, eachdoor assembly (revolving doors 622, 624) preferably includes slottedoptical sensors (not shown) to indicate door open and door closedpositions. Sensors available from Optek Technology, Inc. of Carrollton,Tex., Model No. OPB980T11, are preferred because of the relatively fineresolution provided thereby to permit accurate monitoring of doorposition. A skewed disk linear mixer (also known as a wobbler plate) 634is provided within housing 610 adjacent MTU carousel assembly 671 andoperates as a receptacle mixing mechanism. The mixer 634 comprises adisk mounted in a skewed manner to the shaft of a motor 636 whichextends through opening 635 into the housing 610. The motor ispreferably a VEXTA stepper motor, Model No. PK264-01A, available fromOriental Motors Ltd. of Tokyo, Japan, which is the same motor preferablyused for the MTU carousel assembly 671. A viscous harmonic damper 638 ispreferably attached to motor 636 to damp out harmonic frequencies of themotor which can cause the motor to stall. Preferred harmonic dampers areVEXTA harmonic dampers, available from Oriental Motors Ltd. Theoperation of the skewed disk linear mixer 634 will be described below.

Only two of the incubators, the AMP incubator 604 and the HYB incubator606, include a skewed disk linear mixer 634, because, in the preferredmode of operation, it is only in these two incubators where fluids aredispensed into the MTUs 160 while they are in the incubator. Thus, it isonly necessary to provide linear mixing of the MTU 160 by the skeweddisk linear mixer 634 in the AMP incubator 604 and the HYB incubator606.

To effect linear mixing of an MTU 160 in the incubator by linear mixer634, the MTU carousel assembly 671 moves the MTU 160 into alignment withthe skewed disk linear mixer 634, and the skewed disk of the skewed disklinear mixer 634 engages the MTU manipulating structure 166 of the MTU160. As the motor 636 spins the skewed disk of the skewed disk linearmixer 634, the portion of the skewed disk structure engaged with the MTU160 moves radially in and out with respect to the wall of the housing610, thus alternately engaging the vertical piece 167 of the MTUmanipulating structure 166 and the shield structure 169. Accordingly,the MTU 160 engaged with the skewed disk linear mixer 634 is movedradially in and out, preferably at high frequency, providing linearmixing of the contents of the MTU 160. For the amplification incubationstep of the preferred mode of operation, which occurs within the AMPincubator 604, a mixing frequency of 10 Hz is preferred. For the probeincubation step of the preferred mode of operation, which occurs withinthe HYB incubator 606, a mixing frequency of 14 Hz is preferred.Finally, for the select incubation step of the preferred mode ofoperation, which also occurs within the HYB incubator 606, a mixingfrequency of 13 Hz is preferred.

The raised arcuate portions 171, 172 may be provided in the middle ofthe convex surfaces of the vertical piece 167 and the shield structure169 of the MTU 160, respectively, (see FIG. 60) to minimize the surfacecontact between the skewed disk linear mixer 634 and the MTU 160 so asto minimize friction between the MTU 160 and the skewed disk linearmixer 634.

In the preferred embodiment, a sensor is provided at the skewed disklinear mixer 634 to ensure that the skewed disk linear mixer 634 stopsrotating in the “home” position shown in FIG. 21, so that MTUmanipulating structure 166 can engage and disengage from the skewed disklinear mixer 634 as the MTU carousel assembly 671 rotates. The preferred“home” sensor is a pin extending laterally from the skewed disk linearmixer structure and a slotted optical switch which verifies orientationof the skewed disk linear mixer assembly when the pin interrupts theoptical switch beam. Hall effect sensors based on magnetism may also beused.

An alternate MTU carousel assembly and carousel drive mechanism areshown in FIGS. 23A and 23C. As shown in FIG. 23A, the alternateincubator includes a housing assembly 1650 generally comprising acylindrical portion 1610 constructed of nickel-plated cast aluminum, acover 1676 preferably formed of machined aluminum, insulation 1678 forthe cover 1676, and an insulation jacket 1651 surrounding thecylindrical portion 1610. As with the previously described incubatorembodiment, the incubator may include a linear mixer mechanism includinga linear mixer motor 636 with a harmonic damper 638. A closure mechanism1600 (described below) operates to close off or permit access through areceptacle access opening 1614. As with the previously describedembodiment, the incubator may include one or two access openings 1614depending on the location of the incubator and its function within theanalyzer 50.

A centrifugal fan 632 is mounted at a bottom portion of the housing 1650and is driven by a motor (not shown). A fan cover 1652 is disposed overthe fan and includes sufficient openings to permit air flow generated bythe fan 632. A carousel support shaft 1654 includes a lower shaft 1692and an upper shaft 1690 divided by a support disk 1694. The supportshaft 1654 is supported by means of the lower shaft 1692 extending downinto the fan cover 1652 where it is rotatably supported and secured bybearings (not shown).

An MTU carousel 1656 includes an upper disk 1658 having a centralportion 1696. A top surface of the support disk 1694 engages and isattached to a bottom surface of the central portion 1696 of the upperdisk 1658 so that the weight of the carousel 1656 is supported frombelow. As shown in FIG. 23C, a plurality of radially extending,circumferentially spaced station dividers 1660 are attached beneath theupper disk 1658. A lower disk 1662 includes a plurality of radialflanges 1682 emanating from an annular inner portion 1688. The radialflanges 1682 correspond in number and spacing to the carousel stationdividers 1660, and the lower disk 1662 is secured to the bottom surfacesof the carousel station dividers 1660, with each flange 1682 beingsecured to an associated one of the dividers 1660.

The radial flanges 1682 define a plurality of radial slots 1680 betweenadjacent pairs of flanges 1682. As can be appreciated from FIG. 23C, thewidth in the circumferential direction of each flange 1682 at an innerend 1686 thereof is less than the width in the circumferential directionof the flange 1682 at the outer end 1684 thereof. The tapered shape ofthe flanges 1682 ensures that the opposite sides of the slots 1680 aregenerally parallel to one another.

When the lower disk 1662 is attached beneath the carousel stationdividers 1660, the widths of the flanges along at least a portion oftheir respective lengths are greater than the widths of the respectivedividers 1660, which may also be tapered from an outer end thereoftoward an inner end thereof. The flanges 1684 define lateral shelvesalong the sides of adjacent pairs of dividers 1660 for supporting theconnecting rib structure 164 of an MTU 160 inserted into each MTUstation 1663 defined between adjacent pairs of dividers 1660.

A pulley 1664 is secured to the top of the central portion 1696 of theupper disk 1658 and a motor 1672 is carried by a mounting bracket 1670which spans the diameter of the housing 1650 and is secured to thecylindrical portion 1610 of the housing at opposite ends thereof. Themotor is preferably a Vexta PK264-01A stepper motor, and it is coupledto the pulley (having a 9:1 ratio with respect to the motor) by a belt1666, preferably one supplied by the Gates Rubber Company. A positionencoder 1674 is secured to a top central portion of the mounting bracket1672 and is coupled with the upper shaft 1690 of the carousel supportshaft 1654. The encoder 1674 (preferably an absolute encoder of the A2series by U.S. Digital Corporation of Vancouver, Wash.) indicates therotational position of the carousel 1656.

An incubator cover is defined by an incubator plate 1676, preferablyformed of machined aluminum, and a conforming cover insulation element1678. Cover plate 1676 and insulation element 1678 include appropriateopenings to accommodate the encoder 1674 and the motor 1672 and may alsoinclude radial slots formed therein for dispensing fluids into MTUscarried within the incubator as described with regard to the aboveembodiment.

An alternate, and preferred, closure mechanism 1600 is shown in FIG.23B. The cylindrical portion 1610 of the incubator housing includes atleast one receptacle access opening 1614 with outwardly projecting wallportions 1616, 1618 extending integrally from the cylindrical portion1610 along opposite sides of the access opening 1614.

A rotating door 1620 is operatively mounted with respect to the accessopening 1614 by means of a door mounting bracket 1636 attached to thecylindrical portion 1610 of the housing above the access opening 1614.Door 1620 includes an arcuate closure panel 1622 and a transverselyextending hinge plate portion 1628 having a hole 1634 for receiving amounting post (not shown) of the door mounting bracket 1636. The door1622 is rotatable about the opening 1634 with respect to the accessopening 1614 between a first position in which the arcuate closure panel1622 cooperates with the projecting wall portions 1616, 1618 to closeoff the access opening 1614 and a second position rotated outwardly withrespect to the access opening 1614 to permit movement of a receptaclethrough the access opening 1614. An inner arcuate surface of the arcuatepanel 1622 conforms with an arcuate surface 1638 of the door mountingbracket 1636 and an arcuate surface 1619 disposed below the receptacleaccess opening 1614 to permit movement of the arcuate panel 1622 withrespect to the surfaces 1638 and 1619 while providing a minimum gapbetween the respective surfaces so as to minimize heat losstherethrough.

The door 1620 is actuated by a motor 1642 mounted to the incubatorhousing by means of a motor mounting bracket 1640 secured to thecylindrical portion 1610 of the housing beneath the receptacle accessopening 1614. A motor shaft 1644 is coupled to a lower actuating plate1626 of the rotating door 1620 so that rotation of the shaft 1644 istransmitted into rotation of the rotating door 1620. Motor 1642 is mostpreferably an HIS 7.5° per step motor available from Haydon Switch andInstrument, Inc. of Waterbury, Conn. The HIS motor is chosen because ofits relatively low cost and because the closure assembly 1600 does notrequire a high torque, robust motor.

Door position sensors 1646 and 1648 (preferably slotted optical sensors)are operatively mounted on opposite sides of the door mounting bracket1636. The sensor 1646 and 1648 cooperate with sensor tabs 1632 and 1630on the hinge plate 1628 of the door 1620 for indicating the relativeposition of the rotating door 1620 and can be configured so as toindicate, for example, a door open and a door closed status.

A door cover element 1612 is secured to the outside of the cylindricalportion 1610 of the housing so as to cover the door mounting bracket1636 and a portion of the rotating door 1620. The cover element 1612includes an access opening 1613 aligned with the access opening 1614 ofthe incubator housing and further includes a receptacle bridge 1615extending laterally from a bottom edge of the access opening 1613. Thereceptacle bridge 1615 facilitates the insertion of a receptacle (e.g.,an MTU 160) into and withdrawal of the receptacle from the incubator.

While in the TC incubator 600, the MTU 160 and test samples arepreferably kept at a temperature of about 60° C.±0.5° C. for a period oftime sufficient to permit hybridization between capture probes andtarget nucleic acids. Under these conditions, the capture probes willpreferably not hybridize with those polynucleotides directly immobilizedon the magnetic particles.

Following target capture incubation in the TC incubator 600, the MTU 160is rotated by the incubator carousel to the entrance door 622, alsoknown as the right-side or number one distributor door. The MTU 160 isretrieved from its MTU station 676 within the TC incubator 600 and isthen transferred by the right-side transport mechanism 500 to atemperature ramp-down station (not shown) below the sample ring 250. Inthe ramp-down station, the MTU temperature is brought down to the levelof the next incubator. This ramp-down station that precedes the ATincubator 602 is technically a heater, as opposed to a chiller, becausethe temperature to which the MTU is decreased, about 40° C., is stillgreater than the ambient analyzer temperature, about 30° C. Accordingly,this ramp-down station preferably uses resistive heating elements, asopposed to a thermoelectric module.

From the ramp-down station, the MTU 160 is transferred by the right-sidetransfer mechanism 500 into the AT incubator 602. The design andoperation of the AT incubator 602 is similar to that of the TC incubator600, as described above, except that the AT incubator 602 incubates at40±1.0° C.

In the AT incubator 602, the hybridization conditions are such that thepolythymidine (“poly(dT)”) tail of the immobilized polynucleotide canhybridize to the polyadenine (“poly(dA)”) tail of the capture probe.Provided target nucleic acid has hybridized with the capture probe inthe TC incubator 600, a hybridization complex can be formed between theimmobilized polynucleotide, the capture probe and the target nucleicacid in the AT incubator 602, thus immobilizing the target nucleic acid.

During active temperature binding incubation, the carousel assembly 1656(or 671) of the AT incubator 602 rotates the MTU to the exit door 624,also known as the number two, or left-side, distributor door, from whichthe MTU 160 can be removed by the left-side transport mechanism 502. Theleft-side transport mechanism 502 removes the MTU 160 from the ATincubator 602 and places it into an available magnetic separationstation 800.

Temperature ramping stations 700 can be a bottle neck in the processingof a number of MTUs through the chemistry deck 200. It may be possibleto use underutilized MTU stations 676 in one or more of the incubatorsin which temperature sensitivity is of less concern. For example, theactive temperature binding process which occurs within the AT incubator602 at about 40° C. is not as temperature sensitive as the otherincubators, and up to fifteen (15) of the incubator's thirty (30) MTUstations 676 may be unused at any given time. As presently contemplated,the chemistry deck has only about eight ramp-up stations, or heaters.Accordingly, significantly more MTUs can be preheated within the unusedslots of the AT incubator 602 than within the ramp-up stations 700.Moreover, using unused incubator slots instead of heaters allows theomission of some or all of the heaters, thus freeing up space on thechemistry deck.

Magnetic Separation Stations

Turning to FIGS. 24-25, each magnetic separation station 800 includes amodule housing 802 having an upper section 801 and a lower section 803.Mounting flanges 805, 806 extend from the lower section 803 for mountingthe magnetic separation stations 800 to the datum plate 82 by means ofsuitable mechanical fasteners. Locator pins 807 and 811 extend from thebottom of lower section 803 of housing 802. Pins 807 and 811 registerwith apertures (not shown) formed in the datum plate 82 to help tolocate the magnetic separation stations 800 on the datum plate 82 beforethe housing 802 is secured by fasteners.

A loading slot 804 extends through the front wall of the lower section803 to allow a transport mechanism (e.g. 502) to place an MTU 160 intoand remove an MTU 160 from the magnetic separation station 800. Atapered slot extension 821 surrounds a portion of the loading slot 804to facilitate MTU insertion through the slot 804. A divider 808separates the upper section 801 from the lower section 803.

A pivoting magnet moving structure 810 is attached inside the lowersection 803 so as to be pivotable about point 812. The magnet movingstructure 810 carries permanent magnets 814, which are positioned oneither side of an MTU slot 815 formed in the magnet moving structure810. Preferably five magnets, one corresponding to each individualreaction tube 162 of the MTU 160, are held in an aligned arrangement oneach side of the magnet moving structure 810. The magnets are preferablymade of neodymium-iron-boron (NdFeB), minimum grade—35 and havepreferred dimensions of 0.5 inch width, 0.3 inch height, and 0.3 inchdepth. An electric actuator, generally represented at 816, pivots themagnet moving structure 810 up and down, thereby moving the magnets 814.As shown in FIG. 25, actuator 816 preferably comprises a rotary steppermotor 819 which rotates a drive screw mechanism coupled to the magnetmoving structure 810 to selectively raise and lower the magnet movingstructure 810. Motor 819 is preferably an HIS linear stepper actuator,Model No. 26841-05, available from Haydon Switch and Instrument, Inc. ofWaterbury, Conn.

A sensor 818, preferably an optical slotted sensor, is positioned insidethe lower section 803 of the housing for indicating the down, or “home”,position of the magnet moving structure 810. Sensor 818 is preferably anOptek Technology, Inc., Model No. OPB980T11, available from OptekTechnology, Inc. of Carrollton, Tex. Another sensor 817, also preferablyan Optek Technology, Inc., Model No. OPB980T11, optical slotted sensor,is preferably provided to indicate the up, or engaged, position of themagnet moving structure 810.

An MTU carrier unit 820 is disposed adjacent the loading slot 804, belowthe divider 808, for operatively supporting an MTU 160 disposed withinthe magnetic separation stations 800. Turning to FIG. 26, the MTUcarrier unit 820 has a slot 822 for receiving the upper end of an MTU160. A lower fork plate 824 attaches to the bottom of the carrier unit820 and supports the underside of the connecting rib structure 164 ofthe MTU 160 when slid into the carrier unit 820 (see FIGS. 27 and 28). Aspring clip 826 is attached to the carrier unit 820 with its opposedprongs 831, 833 extending into the slot 822 to releasably hold the MTUwithin the carrier unit 820.

An orbital mixer assembly 828 is coupled to the carrier unit 820 fororbitally mixing the contents of an MTU held by the MTU carrier unit820. The orbital mixer assembly 828 includes a stepper motor 830 mountedon a motor mounting plate 832, a drive pulley 834 having an eccentricpin 836, an idler pulley 838 having an eccentric pin 840, and a belt 835connecting drive pulley 834 with idler pulley 838. Stepper motor 830 ispreferably a VEXTA, Model No. PK245-02A, available from Oriental MotorsLtd. of Tokyo, Japan, and belt 835 is preferably a timing belt, ModelNo. A 6G16-170012, available from SDP/SI of New Hyde Park, N.Y. As shownin FIGS. 25 and 26, eccentric pin 836 fits within a slot 842 formedlongitudinally in the MTU carrier unit 820. Eccentric pin 840 fitswithin a circular aperture 844 formed in the opposite end of MTU carrierunit 820. As the motor 830 turns the drive pulley 834, idler pulley 838also rotates via belt 835 and the MTU carrier unit 820 is moved in ahorizontal orbital path by the eccentric pins 836, 840 engaged with theapertures 842, 844, respectively, formed in the carrier unit 820. Therotation shaft 839 of the idler pulley 838 preferably extends upwardlyand has a transverse slot 841 formed therethrough. An optical slottedsensor 843 is disposed at the same level as the slot 841 and measuresthe frequency of the idler pulley 838 via the sensor beam intermittentlydirected through slot 841 as the shaft 839 rotates. Sensor 843 ispreferably an Optek Technology, Inc., Model No. OPB980T11, sensor,available from Optek Technology, Inc. of Carrollton, Tex.

Drive pulley 834 also includes a locator plate 846. Locator plate 846passes through slotted optical sensors 847, 848 mounted to a sensormounting bracket 845 extending from motor mounting plate 832. Sensors847, 848 are preferably Optek Technology, Inc., Model No. OPB980T11,sensors, available from Optek Technology, Inc. of Carrollton, Tex.Locator plate 846 has a plurality of circumferentially spaced axialopenings formed therein which register with one or both sensors 847, 848to indicate a position of the orbital mixer assembly 828, and thus aposition of the MTU carrier unit 820.

Returning to FIG. 24, wash solution delivery tubes 854 connect tofittings 856 and extend through a top surface of the module housing 802.Wash solution delivery tubes 854 extend through the divider 808 viafittings 856, to form a wash solution delivery network.

As shown in FIGS. 27 and 28, wash solution dispenser nozzles 858extending from the fittings 856 are disposed within the divider 808.Each nozzle is located above a respective reaction tube 162 of the MTU160 at a laterally off-center position with respect to the reaction tube162. Each nozzle includes a laterally-directed lower portion 859 fordirecting the wash solution into the respective reaction tube from theoff-center position. Dispensing fluids into the reaction tubes 162 in adirection having a lateral component can limit splashing as the fluidruns down the sides of the respective reaction tubes 162. In addition,the laterally directed fluid can rinse away materials clinging to thesides of the respective reaction tubes 162.

As shown in FIGS. 24 and 25, aspirator tubes 860 extend through a tubeholder 862, to which the tubes 860 are fixedly secured, and extendthrough openings 861 in the divider 808. A tube guide yoke 809 (see FIG.26) is attached by mechanical fasteners to the side of divider 808,below openings 861. Aspirator hoses 864 connected to the aspirator tubes860 extend to the vacuum pump 1162 (see FIG. 52) within the analyzer 50,with aspirated fluid drawn off into a fluid waste container carried inthe lower chassis 1100. Each of the aspirator tubes 860 has a preferredlength of 12 inches with an inside diameter of 0.041 inches.

The tube holder 862 is attached to a drive screw 866 actuated by a liftmotor 868. Lift motor 868 is preferably a VEXTA, Model No. PK245-02A,available from Oriental Motors Ltd. of Tokyo, Japan, and the drive screw866 is preferably a ZBX series threaded anti-backlash lead screw,available from Kerk Motion Products, Inc. of Hollis, N.H. The tubeholder 862 is attached to a threaded sleeve 863 of the drive screw 866.Rod 865 and slide rail 867 function as a guide for the tube holder 862.Z-axis sensors 829, 827 (slotted optical sensors) cooperate with a tabextending from threaded sleeve 863 to indicate top and bottom of strokepositions of the aspirator tubes 860. The Z-axis sensors are preferablyOptek Technology, Inc., Model No. OPB980T11, sensors, available fromOptek Technology, Inc. of Carrollton, Tex.

Cables bring power and control signals to the magnetic separationstations 800, via a connector 870.

The magnet moving structure 810 is initially in a down position (shownin phantom in FIG. 25), as verified by the sensor 818, when the MTU 160is inserted into the magnetic separation stations 800 through the insertopening 804 and into the MTU carrier unit 820. When the magnet movingstructure 810 is in the down position, the magnetic fields of themagnets 814 will have no substantial effect on the magneticallyresponsive particles contained in the MTU 160. In the present context,“no substantial effect” means that the magnetically responsive particlesare not drawn out of suspension by the attraction of the magnetic fieldsof the magnets 814. The orbital mixer assembly 828 moves the MTU carrierunit 820 a portion of a complete orbit so as to move the carrier unit820 and MTU 160 laterally, so that each of the tiplets 170 carried bythe tiplet holding structures 176 of the MTU 160 is aligned with each ofthe aspiration tubes 860, as shown in FIG. 28. The position of the MTUcarrier unit 820 can be verified by the locator plate 846 and one of thesensors 847, 848. Alternatively, the stepper motor 830 can be moved aknown number of steps to place the MTU carrier unit 820 in the desiredposition, and one of the sensors 847, 848 can be omitted.

The tube holder 862 and aspirator tubes 860 are lowered by the liftmotor 868 and drive screw 866 until each of the aspirator tubes 860frictionally engages a tiplet 170 held in an associated carryingstructure 176 on the MTU 160.

As shown in FIG. 25A, the lower end of each aspirator tube 860 ischaracterized by a tapering, step construction, whereby the tube 860 hasa first portion 851 along most of the extent of the tube, a secondportion 853 having a diameter smaller than that of the first portion851, and a third portion 855 having a diameter smaller than that of thesecond portion 853. The diameter of the third portion 855 is such as topermit the end of the tube 860 to be inserted into the flared portion181 of the through hole 180 of the tiplet 170 and to create aninterference friction fit between the outer surface of third portion 855and the two annular ridges 183 (see FIG. 59) that line the inner wall ofhole 180 of tiplet 170. An annular shoulder 857 is defined at thetransition between second portion 853 and third portion 855. Theshoulder 857 limits the extent to which the tube 860 can be insertedinto the tiplet 170, so that the tiplet can be stripped off after use,as will be described below.

The tiplets 170 are at least partially electrically conductive, so thatthe presence of a tiplet 170 on an aspirator tube 860 can be verified bythe capacitance of a capacitor comprising the aspirator tubes 860 as onehalf of the capacitor and the surrounding hardware of the magneticseparation stations 800 as the other half of the capacitor. Thecapacitance will change when the tiplets 170 are engaged with the endsof the aspirator tubes 860.

In addition, five optical slotted sensors (not shown) can bestrategically positioned above the divider 808 to verify the presence ofa tiplet 170 on the end of each aspirator tube 860. Preferred“tiplet-present” sensors are Optek Technology, Inc., Model No.OPB930W51, sensors, available from Optek Technology, Inc. of Carrollton,Tex. A tiplet 170 on the end of an aspirator tube 860 will break thebeam of an associated sensor to verify presence of the tiplet 170. If,following a tiplet pick-up move, tiplet engagement is not verified bythe tiplet present sensors for all five aspirator tubes 860, the MTU 160must be aborted. The aborted MTU is retrieved from the magneticseparation stations 800 and sent to the deactivation queue 750 andultimately discarded.

After successful tiplet engagement, the orbital mixer assembly 828 movesthe MTU carrier unit 820 back to a fluid transfer position shown in FIG.27 as verified by the locator plate 846 and one or both of the sensors847, 848.

The magnet moving structure 810 is then raised to the up position shownin FIG. 24 so that the magnets 814 are disposed adjacent opposite sidesof the MTU 160. With the contents of the MTU subjected to the magneticfields of the magnets 814, the magnetically responsive particles boundindirectly to the target nucleic acids will be drawn to the sides of theindividual reaction tubes 162 adjacent the magnets 814. The remainingmaterial within the reaction tubes 162 should be substantiallyunaffected, thereby isolating the target nucleic acids. The magnetmoving structure 810 will remain in the raised position for anappropriate dwell time, as defined by the assay protocol and controlledby the assay manager program, to cause the magnetic particles to adhereto the sides of the respective reaction tubes 162.

The aspirator tubes are then lowered into the reaction tubes 162 of theMTU 160 to aspirate the fluid contents of the individual reaction tubes162, while the magnetic particles remain in the reaction tubes 162,adhering to the sides thereof, adjacent the magnets 814. The tiplets 170at the ends of the aspirator tubes 860 ensure that the contents of eachreaction tube 162 do not come into contact with the sides of theaspirator tubes 860 during the aspirating procedure. Because the tiplets170 will be discarded before a subsequent MTU is processed in themagnetic separation stations 800, the chance of cross-contamination bythe aspirator tubes 860 is minimized.

The electrically conductive tiplets 170 can be used in a known mannerfor capacitive fluid level sensing within the reaction tubes 162 of theMTUs. The aspirator tubes 860 and the conductive tiplets 170 compriseone half of a capacitor, the surrounding conductive structure within theparticles comprises the second half of the capacitor, and the fluidmedium between the two halves of the capacitor constitutes thedielectric. Capacitance changes due to a change in the nature of thedielectric can be detected.

The capacitive circuitry of the aspirator tubes 860 can be arranged sothat all five aspirator tubes 860 operate as a single gang level-sensingmechanism. As a gang level-sensing mechanism, the circuitry will onlydetermine if the fluid level in any of the reaction tubes 162 is high,but cannot determine if the fluid level in one of the reaction tubes islow. In other words, when any of the aspirator tubes 860 and itsassociated tiplet 170 contacts fluid material within a reaction tube,capacitance of the system changes due to the change in the dielectric.If the Z-position of the aspirator tubes 860 at which the capacitancechange occurs is too high, then a high fluid level in at least onereaction tube is indicated, thus implying an aspiration failure. On theother hand, if the Z-position of the aspirator tubes at which thecapacitance change occurs is correct, the circuitry cannot differentiatebetween aspirator tubes, and, therefore, if one or more of the othertubes has not yet contacted the top of the fluid, due to a low fluidlevel, the low fluid level will go undetected.

Alternatively, the aspirator tube capacitive circuitry can be arrangedso that each of the five aspirator tubes 860 operates as an individuallevel sensing mechanism.

With five individual level sensing mechanisms, the capacitive levelsensing circuitry can detect failed fluid aspiration in one or more ofthe reaction tubes 162 if the fluid level in one or more of the reactiontubes is high. Individual capacitive level sensing circuitry can detectfailed fluid dispensing into one or more of the reaction tubes 162 ifthe fluid level in one or more of the reaction tubes is low.Furthermore, the capacitive level sensing circuitry can be used forvolume verification to determine if the volume in each reaction tube 162is within a prescribed range. Volume verification can be performed bystopping the descent of the aspirator tubes 860 at a position aboveexpected fluid levels, e.g. 110% of expected fluid levels, to make surenone of the reaction tubes has a level that high, and then stopping thedescent of the aspirator tubes 860 at a position below the expectedfluid levels, e.g. 90% of expected fluid levels, to make sure that eachof the reaction tubes has a fluid level at least that high.

Following aspiration, the aspirator tubes 860 are raised, the magnetmoving structure 810 is lowered, and a prescribed volume of washsolution is dispensed into each reaction tube 162 of the MTU 160 throughthe wash solution dispenser nozzles 858. To prevent hanging drops ofwash solution on the wash solution dispenser nozzles 858, a brief,post-dispensing air aspiration is preferred.

The orbital mixer assembly 828 then moves the MTU carriers 820 in ahorizontal orbital path at high frequency to mix the contents of the MTU160. Mixing by moving, or agitating, the MTU in a horizontal plane ispreferred so as to avoid splashing the fluid contents of the MTU and toavoid the creation of aerosols. Following mixing, the orbital mixerassembly 828 stops the MTU carrier unit 820 at the fluid transferposition.

To further purify the targeted nucleic acids, the magnet movingstructure 810 is again raised and maintained in the raised position fora prescribed dwell period. After magnetic dwell, the aspirator tubes 860with the engaged tiplets 170 are lowered to the bottoms of the reactiontubes 162 of the MTU 160 to aspirate the test sample fluid and washsolution in an aspiration procedure essentially the same as thatdescribed above.

One or more additional wash cycles, each comprising a dispense, mix,magnetic dwell, and aspirate sequence, may be performed as defined bythe assay protocol. Those skilled in the art of nucleic acid-baseddiagnostic testing will be able to determine the appropriate magneticdwell times, number of wash cycles, wash solutions, etc. for a desiredtarget capture procedure.

While the number of magnetic separation stations 800 can vary, dependingon the desired throughput, analyzer 50 preferably includes five magneticseparation stations 800, so that a magnetic separation wash procedurecan be performed on five different MTUs in parallel.

After the final wash step, the magnet moving structure 810 is moved tothe down position and the MTU 160 is removed from the magneticseparation stations 800 by the left-side transport mechanism 502 and isthen placed into the left orbital mixer 552.

After the MTU 160 is removed from the wash station, the tiplets 170 arestripped from the aspiration tubes 860 by a stripper plate 872 locatedat the bottom of the lower section 803 of the housing 802.

The stripper plate 872 has a number of aligned stripping holes 871corresponding in number to the number of aspiration tubes 860, which isfive in the preferred embodiment. As shown in FIGS. 29A to 29D, eachstripping hole 871 includes a first portion 873, a second portion 875smaller than first portion 873, and a bevel 877 surrounding portions 873and 875. The stripper plate 872 is oriented in the bottom of the housing802 so that the small portion 875 of each stripping hole 871 isgenerally aligned with each associated aspiration tube 860, as shown inFIG. 29A. The aspiration tubes 860 are lowered so that the tiplet 170 atthe end of each aspirator tube 860 engages the stripping hole 871. Smallportion 875 is too small to accommodate the diameter of a tiplet 170, sothe bevel 877 directs the tiplet 170 and the aspirator tube 860 towardthe larger portion 873, as shown in FIG. 29B. The aspirator tubes 860are made of an elastically flexible material, preferably stainlesssteel, so that, as the aspirator tubes 860 continue to descend, thebeveled portion 877 causes each of aspirator tubes 860 to deflectlaterally. The small portion 875 of the stripping hole 871 canaccommodate the diameter of the aspirator tube 860, so that after therim 177 of the tiplet 170 clears the bottom of stripping hole 871, eachof the aspirator tubes 860 snaps, due to its own resilience, into thesmall portion 875 of the stripping hole 871 as shown in FIG. 29C. Theaspirator tubes 860 are then raised, and the rim 177 of each tiplet 170engages the bottom peripheral edge of the small portion 875 of strippinghole 871. As the aspirator tubes 860 ascend further, the tiplets 170 arepulled off the aspirator tubes 860 by the stripping holes 871 (see FIG.29D). The stripped tiplets 170 are directed by a chute into a solidwaste container, such as the tiplet waste bin 1134.

The capacitance of the aspiration tubes 860 is sampled to verify thatall tiplets 170 have been stripped and discarded. The stripping step canbe repeated if necessary.

An alternate stripper plate 882 is shown in FIGS. 31A to 31C. Stripperplate 882 includes a number of stripping holes 881 corresponding to thenumber of aspirator tubes 860, which is five in the preferredembodiment. Each stripping hole 881 includes a through-hole 883surrounded by a bevelled countersink 887. A pair of tangs 885 extendlaterally from diametrically opposed positions below the through-hole883. Tangs 885 are preferably made from a spring steel and include av-notch 886 at their ends.

As an aspirator tube 860 with a tiplet 170 disposed on its end islowered toward stripping hole 881, bevelled portion 887 ensures that anymisaligned tubes are directed into the through-hole 883. The spacingbetween the ends of the opposed tangs 885 is less than the diameter ofthe tiplet 170, so as the aspirator tube 860 and tiplet 170 are lowered,the tiplet engages the tangs 885, causing them to deflect downwardly asthe tiplet 170 is forced between tangs 885. When the aspirator tubes 860are raised, the notches 886 of the tangs 885 grip the relatively softmaterial of the tiplet 170, thus preventing upward relative movement ofthe tiplet 170 with respect to the tangs 885. As the tubes continue toascend, the tangs 885 pull the tiplet 170 off the tube 860. When theaspirator tubes 860 are subsequently lowered to strip a subsequent setof tiplets, the tiplet held between the tangs from the previousstripping is pushed through the tangs by the next tiplet and is directedtoward waste bin 1134 (see FIG. 52) located in the lower chassis 1100generally below the five magnetic separation stations 800.

Still another alternate, and the presently preferred, stripper plate1400 is shown in FIGS. 30A-30D. Stripper plate 1400 includes fivestripper cavities 1402, each including an initial frusto-conical portion1404. The frusto-conical portion 1404 tapers down to a neck portion 1406which connects to an enlarged straight section 1408. Straight section1408 is offset with respect to the center of neck portion 1406, so thatone side of the straight section 1408 is flush with a side of the neckportion 1406, and an opposite side of the straight section 1408 isoffset from and undercuts the side of the neck portion 1406, therebyforming a ledge 1414. Following the straight section 1408, a slopedportion 1410 is provided on a side of the stripper cavity 1402 oppositethe ledge 1414. Sloped portion 1410 tapers inwardly toward a bottomopening 1412.

As an aspirator tube 860 with a tiplet 170 on its end is moved towardthe stripper cavity 1402, the frusto-conical portion 1404 directs thetiplet 170 and tube 860 toward the neck portion 1406. The aspirator tube860 continues to descend, and the tiplet 170 enters the straight section1408 as the rim 177 of the tiplet 170 clears the bottom of thefrusto-conical portion 1404 and passes through the neck portion 1406.

If the aspirator tube 860 and the stripper cavity 1402 are in proper,preferred alignment, a portion of the rim 177 of the tiplet 170 will bedisposed below the ledge 1414 of the stripper cavity 1402 when thetiplet 170 has moved through the neck portion 1406 and into the straightsection 1408. To ensure that a portion of the rim 177 will be disposedbeneath the ledge 1414, the tiplet 170 engages the lower sloped portion1410 as the aspirator tube 860 descends further to urge the aspiratortube laterally to direct the tiplet 170 below the ledge 1414.

The annular shoulder 857 (see FIG. 25A) formed at the bottom of theaspirator tube 860 ensures that the tube 860 is not forced further intothe through hole 180 of the tiplet 170 as the tube 860 is lowered intothe stripper cavity 1402. The aspirator tube 860 then ascends, and theledge 1414 catches the rim 177 and strips the tiplet 170 off the tube860. The stripped tiplet 170 falls through bottom opening 1412 and intothe waist bin 1134 in the lower chassis 1100 (see FIG. 52).

With each of the stripper plates described above, the position of thetiplet-stripping elements are not all the same. For example, the ledges1414 of the stripper cavities 1402 of the stripper plate 1400 are not atthe same height throughout all the cavities. Preferably, threetiplet-stripping elements are at one height, and two tiplet-strippingelements are at a slightly different height above or below the otherthree elements. The result of the offset tiplet-stripping elements isthat the static friction of the tiplet 170 on the end of the aspiratortube 860 need not be overcome, or broken, for all five tubes 860 atonce. As the aspirator tubes 860 begin to ascend, static friction of thetiplets 170 is broken for one set (two or three) of aspirator tubes 860first, and then, as the tubes 860 continue to ascend, static friction ofthe tiplets 170 is broken for the remaining tubes 860. By not breakingstatic friction of the tiplets 170 for all five aspirator tubes 860 atonce, the loads to which the tube holder 862, drive screw 866, threadedsleeve 863, and lift motor 868 are subjected are kept to a lower level.

Orbital Mixers

The left orbital mixer 552 (and the right orbital mixer 550), as shownin FIGS. 32-34, are constructed and operate in the same manner as thelower housing section 803 and the orbital mixer assembly 828 of themagnetic separation stations 800 described above. Specifically, theorbital mixer 550 (552) includes a housing 554, including a front plate551, a back plate 559, and mounting flanges 555, 556, for mounting theorbital mixer 550 (552) to the datum plate 82. An insert opening 557 isformed in a front edge of the housing 554. An MTU carrier 558 has a forkplate 560 attached to the bottom thereof and an MTU-retaining clip 562attached to a back portion of the carrier 558 with opposed prongs of theclip 562 extending into an inner cavity of the carrier 558 thataccommodates the MTU. An orbital mixer assembly 564 includes a drivemotor 566 mounted to a motor mounting plate 567, a drive wheel 568having an eccentric pin 570, an idler wheel 572 having an eccentric pin573, and a belt 574. Drive motor 566 is preferably a stepper motor, andmost preferably a VEXTA, Model No. PK245-02A, available from OrientalMotors Ltd. of Tokyo, Japan. Belt 574 is preferably a timing belt, ModelNo. A 6G16-170012, available from SDP/SI of New Hyde Park, N.Y. Theorbital mixer assembly 564 is coupled to the MTU carrier 558 through theeccentric pins 570, 573 to move the MTU carrier 558 in an orbital pathto agitate the contents of the MTU. The drive wheel 568 includes alocator plate 576, which, in conjunction with sensor 578 attached tosensor mounting bracket 579, verifies the proper positioning of the MTUcarrier 558 for inserting an MTU 160 into the orbital mixer 552 (550)and retrieving an MTU 160 from the orbital mixer. Sensor 578 ispreferably an Optek Technology, Inc., Model No. OPB980T11, sensor,available from Optek Technology, Inc. of Carrollton, Tex.

A top plate 580 is attached atop housing 554. Top plate 580 of the leftorbital mixer 552 includes a number of tube fittings 582, preferablyfive, to which are coupled a like number of flexible delivery tubes (notshown) for delivering a fluid from a bulk fluid container to an MTU 160located within the mixer via dispenser nozzles 583. Top plate 580 alsoincludes a plurality of pipette openings 581, corresponding in number tothe number of individual reaction tubes 162 comprising a single MTU 160,which is preferably five.

With the MTU 160 held stationary in the left orbital mixer 552, pipetteunit 480 of the left pipette assembly 470 transfers a prescribed volumeof amplification reagent from a container within the reagent cooling bay900 into each reaction tube 162 of the MTU 160 through the pipetteopenings 581. The amplification reagent contains at least oneamplification oligonucleotide, such as a primer, a promoter-primer,and/or a promoter oligonucleotide, nucleoside triphosphates, andcofactors, such as magnesium ions, in a suitable buffer. The specificcomponents of the amplification reagent will, however, depend on theamplification procedure being practiced. See, e.g., Kacian et al. inU.S. Pat. No. 5,399,491. Other amplification procedures are well knownto those skilled in the art of nucleic acid-based testing, some of whichare identified supra in the “Background of the Invention” section, andmay be adapted for use in the analyzer 50 of the present invention.

Next, the contents of the MTU are mixed by the orbital mixer assembly564 of the orbital mixer 552 to ensure proper exposure of the targetnucleic acid to amplification reagent. For any particular amplificationprocedure, those skilled in the art will be able to determine theappropriate components and amounts of an amplification reagent, as wellas mix frequencies and durations.

After pipetting amplification reagent into the MTU 160, the pipette unit480 is moved to a rinse basin (described below) on the processing deck200, and pipette unit 480 is washed by running distilled water throughprobe 481. The distilled water is pumped from bottle 1140 in the lowerchassis 1100, and the purge water is collected in a liquid wastecontainer 1128 in the lower chassis 1100.

After mixing the contents of the MTU 160, a layer of silicone oil isdispensed into each reaction tube 162 through the dispenser nozzles 583.The layer of oil, pumped from bottles 1168 in the lower chassis 1100,helps prevent evaporation and splashing of the fluid contents of the MTU160 during subsequent manipulation and incubation of the MTU 160 and itscontents.

Reagent Cooling Bay

The reagent cooling bay 900 will now be described.

Referring to FIGS. 35-39, the reagent cooling bay 900 includes aninsulating jacket 902 fitted around a cylindrical housing 904,preferably made from aluminum. A cover 906, preferably made of Delrin,sits atop housing 904 with a registration tab 905 of cover 906 fittingwithin slot 907 in housing 904 to ensure proper orientation of the cover906. An optical sensor may be provided proximate to or within slot 907for verifying that tab 905 is seated within slot 907. Alternatively, anoptical sensor assembly 909 can be secured to an edge of an upper rim ofthe housing 904 for verifying cover placement. The optical sensorassembly 909 cooperates with a sensor-tripping structure (not shown) onthe cover 906 to verify that the cover is in place. Optical sensorassembly 909 preferably includes an Optek Technology, Inc. slottedoptical sensor, Model No. OPB980T11, available from Optek Technology,Inc. of Carrollton, Tex. The cover 906 also includes pipette openings908 through which pipette units 480, 482 can access reagent containerswithin the cooling bay 900.

The housing 904 is attached to a floor plate 910, and the floor plate910 is attached to the datum plate 82 by means of suitable mechanicalfasteners extending through openings formed in mounting flanges 911spaced about the periphery of the floor plate 910. Cooling units 912,preferably two, are attached to floor plate 910. Each cooling unit 912comprises a thermoelectric module 914 attached cool-side-up to thebottom surface of floor plate 910. Thermoelectric modules available fromMelcor, Inc. of Trenton, N.J., Model No. CP1.4-127-06L, provide thedesired cooling capacity. A heat sink 916, including a plurality ofheat-dissipating fins 915, is attached to, or may be integral with, thebottom surface of floor plate 910, directly below the thermoelectricmodule 914. A fan unit 918 is attached in a position to drain heat awayfrom heat sink 916. Fan units 918 are preferably Orix fans, Model No.MD825B-24, available from Oriental Motors Ltd. of Tokyo, Japan.Together, the cooling units 912 cool the interior of the housing 904 toa prescribed temperature for the benefit of temperature-sensitivereagents (e.g., enzymes) stored within the bay 900.

Two temperature sensors (only one temperature sensor 920 is shown) aredisposed within the cooling bay 900 housing 904 for monitoring andcontrolling the interior temperature thereof. The temperature sensorsare preferably thermistors (10 KOhm at 25° C.), and YSI 44036 seriesthermistors available from YSI, Inc. of Yellow Springs, Ohio are mostpreferred. YSI thermistors are preferred because of their high accuracyand the ±0.1° C. interchangeability provided by YSI thermistors from onethermistor to another. One of the sensors is a primary temperaturecontrol sensor, and the other is a temperature monitoring sensor. On thebasis of the temperature indications from the primary control sensor,the embedded controller adjusts power to the thermoelectric modules 914and/or power to the fan units 918 to control cooling bay temperature.The temperature monitoring sensor provides a verification check of theprimary temperature control sensor.

As shown in FIG. 38, container tray 922 is a one-piece turntablestructure with bottle-holding cavities 924 sized and shaped to receiveand hold specific reagent bottles 925. A drive system for container tray922 includes a motor 926, a small pulley 931 on the shaft of motor 926,a belt 928, a pulley 930, and a shaft 932. (a VEXTA stepper motor, ModelNo. PK265-02A, available from Oriental Motor Co., Ltd. of Tokyo, Japan,and an SDP timing belt, GT® Series, available from SDP/SI of New HydePark, N.Y., are preferred). Motor 926 and cooling units 912 extendthrough openings (not shown) formed in the datum plate 82 and extendbelow the floor plate 910.

Container tray 922 may include a central, upstanding handle 923 tofacilitate installation of the tray 922 into and removal of the tray 922from the housing 904. A top portion 933 of shaft 932 extends throughfloor plate 910 and is received by a mating aperture (not shown) formedin the bottom of the tray 922. A sensor 940 extending up through thefloor plate 910 and into the housing 904 verifies that tray 922 is inplace within the housing 904. Sensor 940 is preferably a capacitiveproximity sensor available from Advanced Controls, Inc., of Bradenton,Fla., Model No. FCP2.

A position encoder 934 (preferably a slotted disk) in conjunction withan optical sensor 935 may be used to detect the position of thecontainer tray 922, so that a specific reagent bottle 925 may be alignedunder the pipette openings 908 in the cover 906.

As shown in FIG. 37, a preferred alternative to the position encoder 934and optical sensor 935 includes four slotted optical sensors 937 (onlytwo sensors are visible in FIG. 36) provided inside the housing 904along with a flag pin (not shown) extending from the bottom of containertray 922. One sensor is provided for each quadrant of the container tray922, and the flag trips one of the four sensors to indicate whichquadrant of the container tray 922 is aligned with the pipette openings908. Sensors 937 are preferably Optek Technology, Inc. sensors, ModelNo. OPB980T11, available from Optek Technology, Inc. of Carrollton, Tex.

A preferred alternative to the one-piece container tray 922 shown inFIG. 38 is a modular tray 1922 shown in FIGS. 35 and 39. Tray 1922includes a circular base plate 1926 and an upstanding handle post 1923attached to a central portion thereof. Modular pieces 1930 havingbottle-holding cavities 1924 are preferably connected to one another andto the base plate 1926 by pins 1928 and screws (not shown) to form thecircular tray 1922. Other means of securing the modular pieces 1930 maybe employed in the alternative to pins 1928 and screws. The modularpieces 1930 shown in the figures are quadrants of a circle, and thus, ofcourse, four such pieces 1930 would be required to complete the tray1922. Although quadrants are preferred, the modular pieces may howeverbe sectors of various sizes, such as, for example, ½ of a circle or ⅛ ofa circle.

Alphanumeric bottle location labels 1940 are preferably provided on thebase plate 1926 to identify positions within the tray 1922 for reagentcontainers. The preferred label scheme includes an encircledletter-number pair comprising a leading letter A, E, P, or S with atrailing number 1, 2, 3, or 4, The letters A, E, P, and S, designateamplification reagent, enzyme reagent, probe reagent, and selectreagent, respectively, corresponding to the preferred mode of use of theanalyzer 50, and the numbers 1-4 designate a quadrant of the tray 1922.Each modular piece 1930 includes a circular hole 1934 at the bottom ofeach bottle-holding cavity 1924. The holes 1934 align with the bottlelocation labels 1940, so that the labels 1940 can be seen when themodular pieces 1930 are in place on the base plate 1926.

The modular pieces 1930 of the container tray 1922 are configured toaccommodate reagent containers of different sizes corresponding toreagent quantities sufficient for performing two hundred fifty (250)assays or reagent quantities sufficient for performing five hundred(500) assays. Four 250-assay modular quadrants permit the reagentcooling bay to be stocked for 1000 assays, and four 500-assay modularquadrants permit the reagent cooling bay to be stocked for 2000 assays.Modular quadrants for 250 or 500 assay reagent kits can be mixed andmatched to configure the container tray for accommodating variousnumbers of a single assay type or various numbers of multiple differentassay types.

An insulation pad 938 is disposed between the container tray 922 and thefloor plate 910. Power, control, temperature, and position signals areprovided to and from the reagent cooling bay 900 by a connector 936 anda cable (not shown) linked to the embedded controller of the analyzer50.

A bar code scanner 941 is mounted to an upstanding scanner mountingplate 939 attached to floor plate 910 in front of an opening 942 formedin a side-wall of the cooling bay 900. The bar code scanner 941 is ableto scan bar code information from each of the reagent containers carriedon the container tray 922. As shown in FIG. 39, longitudinal slots 1932are formed along the bottle-holding cavities 1924, and bar codeinformation disposed on the sides of the reagent container held in thebottle-holding cavities 1924 can be align with the slots 1932 to permitthe bar code scanner 941 to scan the bar code information. A preferredbar code scanner is available from Microscan of Newbury Park, Calif.under Model No. FTS-0710-0001.

Pipette rinse basins 1942, 1944 are attached to the side of the housing904. Each rinse basin 1942, 1944 provides an enclosure structure with aprobe-receiving opening 1941, 1945, respectively, formed in a top panelthereof and a waste drain tube 1946, 1948, respectively, connected to abottom portion thereof. A probe of a pipette unit can be inserted intothe rinse basin 1942, 1944 through the probe-receiving opening 1941,1945, and a wash and/or rinse fluid can be passed through the probe andinto the basin. Fluid in the rinse basin 1942, 1944 is conducted by therespective waste drain tube 1946, 1948 to the appropriate waste fluidcontainer in the lower chassis 1100. In the preferred arrangement andmode of operation of the analyzer 50, probe 481 of pipette unit 480 isrinsed in rinse basin 1942, and probe 483 of pipette unit 482 is rinsedin rinse basin 1944.

After the amplification reagent and oil are added to the reaction tubes162 of MTU 160 in the left orbital mixer 552, the left-side transportmechanism 502 retrieves the MTU 160 from the left orbital mixer 552 andmoves the MTU 160 to an available temperature ramp-up station 700 thatis accessible to the left-side transport mechanism 502, i.e. on theleft-side of the chemistry deck 200, to increase the temperature of theMTU 160 and its contents to about 60° C.

After sufficient ramp-up time in the ramp-up station 700, the left-sidetransport mechanism 502 then moves the MTU 160 to the TC incubator 600.The left-side distributor door 624 of the TC incubator 600 opens, andthe MTU carousel assembly 671 within the TC incubator 600 presents anempty MTU station 676 to permit the left-side transport mechanism toinsert the MTU into the TC incubator 600. The MTU 160 and its contentsare then incubated at about 60° C. for a prescribed incubation period.During incubation, the MTU carousel assembly 671 may continually rotatewithin the TC incubator 600 as other MTUs 600 are removed from andinserted into the TC incubator 600.

Incubating at 60° C. in the TC incubator 600 permits dissociation of thecapture probe/target nucleic acid hybridization complex from theimmobilized polynucleotide present in the assay solution. At thistemperature, an amplification oligonucleotide (e.g., a primer,promoter-primer or promoter oligonucleotide) introduced from the reagentcooling bay 900 can hybridize to the target nucleic acid andsubsequently facilitate amplification of the target nucleotide basesequence.

Following incubation, the MTU carousel assembly 671 within the TCincubator 600 rotates the MTU 160 to the left-side distributor door 624,the left-side distributor door 624 opens, and the left-side transportmechanism 502 retrieves the MTU 160 from the MTU carousel assembly 671of the TC incubator 600. The left-side transport mechanism 502 thenmoves the MTU 160 to, and inserts the MTU 160 into, an availabletemperature ramp-down station 700 that is accessible to the left-sidetransport mechanism 502. The temperature of the MTU 160 and its contentsis decreased to about 40° C. in the ramp-down station. The MTU 160 isthen retrieved from the ramp-down station by the left-side transportmechanism 502 and is moved to the AT incubator 602. The left-sidedistributor door 624 of the AT incubator 602 opens, and the MTU carouselassembly 671 within the AT incubator 602 presents an empty MTU station676, so that the left-side transport mechanism 502 can insert the MTUinto the AT incubator 602. Within the AT incubator 602, the MTU isincubated at about 41° C. for a period of time necessary to stabilizethe temperature of the MTU.

From the AT incubator 602, the MTU is moved by transport mechanism 502to the AMP incubator 604 in which the temperature of the MTU isstabilized at 41.5° C. The MTU carousel assembly 671 within the AMPincubator 604 rotates to place the MTU at the pipetting station belowthe pipette openings 662 formed in the cover 611 (see, e.g., FIG. 19).The container tray 922 within the reagent cooling bay 900 rotates toplace the enzyme reagent container below a pipette opening 908, andpipette unit 482 of pipette assembly 470 transfers an enzyme reagentcontaining one or more polymerases needed for enzymatic synthesis fromthe reagent cooling bay 900 to each of the reaction tubes 162 of the MTU160.

As explained above, pipette units 480, 482 use capacitive level sensingto ascertain fluid level within a container and submerge only a smallportion of the end of the probe 481, 483 of the pipette unit 480, 482 topipette fluid from the container. Pipette units 480, 482 preferablydescend as fluid is drawn into the respective probe 481, 483 to keep theend of the probe submerged to a constant depth. After pipetting reagentinto the pipette unit 480 or 482, the pipette unit creates a minimumtravel air gap of 10 μl in the end of the respective probe 481 or 483 toensure no drips fall from the end of the probe.

After enzyme reagent is added to each reaction tube 162, the MTUcarousel assembly 671 of AMP incubator 604 rotates MTU 160 to the skeweddisk linear mixer 634 within AMP incubator 604 and the MTU 160 and itscontents are mixed as described above at about 10 Hz to facilitateexposure of the target nucleic acid to the added enzyme reagent. Thepipette unit 482 is moved to rinse basin 1942, and the probe 483 isrinsed by passing distilled water through it.

The MTU 160 is then incubated within AMP incubator 604 at about 41.5° C.for a prescribed incubation period. The incubation period should besufficiently long to permit adequate amplification of at least onetarget nucleotide base sequence contained in one or more target nucleicacids which may be present in the reaction tubes 162. Although thepreferred embodiment is designed to facilitate amplification following aTMA procedure, practitioners will easily appreciate those modificationsnecessary to perform other amplification procedures using the analyzer50. In addition, an internal control sequence is preferably added at thebeginning of the assay to provide confirmation that the amplificationconditions and reagents were appropriate for amplification. Internalcontrols are well known in the art and require no further discussionhere. See, e.g., Wang et al., “Quantitation of Nucleic Acids Using thePolymerase Chain Reaction,” U.S. Pat. No. 5,476,774.

Following amplification incubation, the MTU 160 is moved by theleft-side transport mechanism 502 from the AMP incubator 604 to anavailable ramp-up station 700 that is accessible to the left-sidetransport mechanism 502 to bring the temperature of the MTU 160 and itscontents to about 60° C. The MTU 160 is then moved by the left-sidetransport mechanism 502 into the HYB incubator 606. The MTU 160 isrotated to a pipetting station in the HYB incubator 606, and a probereagent from the reagent cooling bay 900 is pipetted into each reactiontube 162, through openings 662 in the cover 611 of the HYB incubator606, by the pipette unit 480. In a preferred embodiment, the probereagent includes a chemiluminescent detection probe, and preferablyacridinium ester (AE)-labeled probe which can be detected in aHybridization Protection Assay (HPA). Acridinium ester-labeled probesand HPA methods are well known in the art. See, e.g., Arnold et al.,U.S. Pat. Nos. 5,639,604, 4,950,613, 5,185,439, and 5,585,481; andCampbell et al., U.S. Pat. No. 4,946,958. While AE-labeled probes andHPA are preferred, the analyzer 50 can be conveniently adapted toaccommodate a variety of detection methods and associated probes, bothlabeled and unlabeled. Confirmation that detection probe has been addedto the reaction tubes 162 can be accomplished using an internal controlthat is able to hybridize (or a complement of the internal control thatis able to hybridize) to a probe in the probe reagent, other than thedetection probe which binds to the target sequence or its complement,under HPA conditions extant in the reaction tubes 162 in the HYBincubator 606. The label of this probe must be distinguishable from thelabel of the detection probe. See, e.g., Nelson et al., “Compositionsand Methods for the Simultaneous Detection and Quantitation of MultipleSpecific Nucleic Acid Sequences,” U.S. Pat. No. 5,827,656.

After dispensing probe reagent into each of the reaction tubes 162 ofthe MTU 160, the pipette unit 480 moves to the pipette rinse basin 1944,and the probe 481 of the pipette unit is rinsed with distilled water.

The MTU carousel assembly 671 rotates the MTU 160 to the skewed disklinear mixer 634 where the MTU 160 and its contents are mixed, asdescribed above, at about 14 Hz to facilitate exposure of amplificationproduct containing the target sequence or its complement to the addeddetection probe. The MTU 160 is then incubated for a period of timesufficient to permit hybridization of the detection probes to the targetsequence or its complement.

After hybridization incubation, the MTU 160 is again rotated within theHYB incubator 606 by the MTU carousel assembly 671 to the pipettingposition below the pipette openings 662. A selection reagent stored in acontainer in the reagent cooling bay 900 is pipetted into each reactiontube 162 by the pipette unit 480. A selection reagent is used with theHPA assay and includes an alkaline reagent that specifically hydrolyzesacridinium ester label which is associated with unhybridized probe,destroying or inhibiting its ability to chemiluminesce, while acridiniumester label associated with probe hybridized to an amplification productcontaining the target sequence or its complement is not hydrolyzed andcan chemiluminesce in a detectable manner under appropriate detectionconditions.

Following addition of the selection reagent to each of the reactiontubes 162 of the MTU 160, the pipette probe 481 of the pipette unit 480is rinsed with distilled water at the pipette rinse basin 1944. The MTU160 is rotated by the MTU carousel assembly 671 within the HYB incubator606 to the skewed disk linear mixer 634 and mixed, as described above,at about 13 Hz to facilitate exposure of the amplification product tothe added selection reagent. The MTU is then incubated in the HYBincubator 606 for a period of time sufficient to complete the selectionprocess.

After selection incubation is complete, the left-side transportmechanism 502 transfers the MTU 160 into an available ramp-down station700 that is accessible to the left-side transport mechanism 502 to coolthe MTU 160. After the MTU 160 is cooled, it is retrieved from theramp-down station by the left-side transport mechanism 502 and is movedby the transport mechanism 502 into the AT incubator 602 to stabilizethe temperature of the MTU 160 at about 40° C.

When a period sufficient to stabilize the temperature of the MTU 160 haspassed, the MTU carousel assembly 671 within AT incubator 602 rotates topresent the MTU 160 at the right-side distributor door of the ATincubator 602. The right-side distributor door 622 is opened and the MTU160 is removed from AT incubator 602 by right-side transport mechanism500.

The right-side transport mechanism 500 moves the MTU to a bar codescanner (not shown) which scans MTU bar code information posted on thelabel-receiving surface 175 of the label-receiving structure 174 of theMTU 160. The bar code scanner is preferably attached to an outer wall ofthe housing of the luminometer 950. A preferred bar code scanner isavailable from Opticon, Inc., of Orangeburg, N.Y., as part numberLHA1127RR1S-032. The scanner verifies the total time of assay prior toentering the luminometer 950 by confirming the correct MTU at thecorrect assaytime. From the bar code reader, the right-side transportmechanism 500 moves the MTU 160 to the luminometer 950.

In a preferred mode of operation, before the right-side transportmechanism 500 moves the MTU 160 into the luminometer 950, the MTU 160 isplaced by the right-side transport mechanism 500 into an available MTUramp-down station, or chiller, to decrease the temperature of the MTU160 to 24±3° C. It has been determined that the MTU contents exhibit amore consistent chemiluminescent “light-off” at this cooler temperature.

Luminometer

Referring to FIGS. 40-42C, a first embodiment of the luminometer 950includes an electronics unit (not shown) within a housing 954. Aphotomultiplier tube (PMT) 956 linked to the electronics unit extendsfrom within the housing 954 through a PMT plate 955, with the front endof the PMT 956 aligned with an aperture 953. A preferred PMT isavailable from Hamamatsu Corp. of Bridgewater, N.J. as Model No. HC 135.Signal measurements using the preferred PMT are based on the well knownphoton counter system.

The aperture 953 is centered in an aperture box 958 in front of the PMTplate 955. The aperture 953 and aperture box 958 are entirely enclosedby a housing, defined by a floor plate 964, a top plate 966, the PMTplate 955, and a back frame 965 and back plate 967, which prevents straylight from entering the aperture 953 and which is attached to the datumplate 82. An MTU transport path extends through the housing in front ofthe aperture 953, generally transversely to an optical axis of theaperture. MTUs 160 pass through the luminometer 950 via the MTUtransport path. A back rail 991 and a front rail 995 are disposed onopposite sides of the MTU transport path and provide parallel horizontalflanges which support the connecting rib structure 164 of an MTU 160disposed within the luminometer 950. Revolving doors 960 are supportedfor rotation within associated door housings 961 disposed on oppositeends of the MTU transport path and are turned by door motors 962, whichmay comprise stepper motors or DC gear motors.

The door housings 961 provide openings through which MTUs 160 can enterand exit the luminometer 950. An MTU 160 enters the luminometer 950 bymeans of the right-side transport mechanism 500 inserting the MTU 160through one of the door housings 961. The MTU 160 exits the luminometerunder the influence of an MTU transport assembly, various embodiments ofwhich are described below, which moves MTUs through the MTU transportpath and eventually out of the luminometer through the other doorhousing 961.

Revolving doors 960 are generally cylindrical and include a cut-outportion 963. Each revolving door 960 can be rotated between an openposition, in which the cut-out portion 963 is generally aligned with theopening of the associated door housing 961, so that an MTU 160 can passthrough the opening, and a closed position, in which a side of therevolving door opposite the cut-out portion 963 extends across theopening of the associated door housing 961 so that neither an MTU 160nor light can pass through the opening. Except when an MTU 160 isentering or exiting the luminometer 950, the revolving doors 960 arepreferably in their respective closed positions to prevent stray lightfrom entering the luminometer. Because test results are ascertained bythe amount of light detected by the PMT 956, stray light from sourcesother than the receptacle 160 being sampled can cause erroneous results.

As shown in FIGS. 40-42C, the MTU transport assembly may include an MTUadvance motor 972 which drives a lead screw 974 through a timing belt(not shown) or bevel gears (not shown). A screw follower 976 engaged tothe lead screw 974 is coupled to an MTU bracket 977 extending away fromlead screw 974 to engage the MTU 160. The MTU bracket 977 has a guideflange 978 with an elongated, slightly arcuate guide hole 979 formedtherein. A guide rod 980 extends through the luminometer 950 adjacentand parallel to the lead screw 974. Guide rod 980 extends through guidehole 979.

To advance the MTU bracket 977 (from bottom to top in FIG. 42C), thelead screw 974 turns counter-clockwise, as viewed in FIG. 42B. Due tosystem friction, the screw follower 976 and the MTU bracket 977 willalso turn counter-clockwise with the lead screw 974 until the guide rod980 contacts the left-side of the guide hole 979. When guide rod 980contacts the side of guide hole 979, MTU bracket 977 and screw follower976 can no longer rotate with lead screw 974, and further rotation ofthe lead screw 974 will cause the MTU bracket 977 and screw follower 976to advance along the lead screw 974. Arms 981 extending from the MTUbracket 977 will also rotate counter-clockwise over a limited arc toengage the MTU 160 and advance it through the luminometer 950, as thelead screw 974 rotates.

After the MTU 160 has passed the PMT 956, that MTU is ejected from theluminometer 950 and the next MTU can be pulled through the luminometer950. The MTU bracket 977 moves toward the MTU entrance end of the MTUtransport path by clockwise rotation of the lead screw 974. Systemfriction will cause the screw follower 976 and MTU bracket 977 to rotateclockwise until the guide rod 980 contacts the right-side of guideopening 979, after which, continued rotation of the lead screw 974 willcause the screw follower 976 and the MTU bracket 977 to retreat alongthe lead screw 974. This clockwise movement of the MTU bracket 977 willcause the arms 981 to rotate clockwise over a limited arc to disengagefrom the MTU, so the MTU bracket 977 can retreat without contacting theMTU. That is, the arms 981 will pass over the top of the MTU as the MTUbracket 977 retreats

As shown in FIG. 41, a blinder 982, driven by a blinder actuator 993,moves vertically up and down, in alignment with the aperture 953.Blinder 982 includes a front panel 983 which is mounted for slidingmovement with respect to the aperture box 958 and which includes agenerally rectangular opening (not shown) formed therein which can bealigned with the aperture 953. A top portion of the front panel 983blocks the aperture 953 when the opening formed in panel 983 is notaligned with the aperture 953 and thus operates as a shutter for theaperture 953. The blinder 982 includes two side-walls 987, arranged inparallel on opposite sides of the opening and generally perpendicular tothe front panel 983, and a back wall 988 spanning the back edges of thesidewalls 987 opposite the front wall 983 and generally parallel to thefront wall 983. The side-walls 987 and the back wall 988 define apartial rectangular enclosure sized to accommodate one reaction tube 162of the MTU 160 when the blinder 982 is moved up beneath one of thereaction tubes 162 of an MTU 160 by the blinder actuator 993. Blinderactuator 993 may be a linear stepper actuator including a stepper motor992 and a lead screw 994. HIS linear stepper actuators, available fromHaydon Switch and Instrument, Inc. of Waterbury, Conn. have been used.

After the MTU 160 is placed into the luminometer 950 by the right-sidetransport mechanism 500, the motor 972 is energized to pull the firstreaction tube of the MTU into alignment with the aperture 953. Theblinder 982, which is normally stowed out of the MTU transport path, israised by the blinder actuator 993 until the side walls 987 and backwall 988 of the blinder 982 surround the reaction tube 162 and theopening formed in the front panel 983 of the blinder 982 is aligned withthe aperture 953. The blinder 982 substantially prevents light fromsources other than the reaction tube 162 in front of the aperture 953from reaching the aperture 953, so that the PMT 956 detects only lightemissions from the reaction tube directly in front of the aperture 953.

With the PMT shutter open, different detection reagents (Detect I andDetect II), drawn from containers 1146, 1170 of the lower chassis 1100,are sequentially delivered into the aligned reaction tube 162 throughdedicated delivery lines (not shown) extending to a reagent port 984 atthe top of the luminometer 950. The Detect I and Detect II reagents arehydrogen peroxide-containing and sodium hydroxide-containing reagents,respectively, and combine to form a basic hydrogen peroxide solutionwhich enhances the chemiluminescence of acridinium ester label which hasnot been hydrolyzed. Because basic hydrogen peroxide is unstable, theDetect I and Detect II reagents are preferably combined in the reactiontube 162 just prior to detection in the luminometer 950.

After the addition of Detect II, the light emitted from the contents ofthe reaction tube 162 is detected using the PMT 956 and the PMT shutteris then closed. The PMT 956 converts light emitted by chemiluminescentlabels into electrical signals processed by the electronics unit andthereafter sent to the controller 1000 or other peripheral unit viacables (not shown) linked to a connector 986.

In cases where less sensitivity is required, it may be possible to usean optical sensor in place of a photomultiplier tube. A diode is anexample of an acceptable optical sensor which can be used with theluminometer 950. An optical sensor may also be appropriate when thematerial of the MTU 160 is relatively transparent, rather than thetranslucent appearance of the preferred polypropylene material. Whenselecting a material for the MTU 160, care should be taken to avoidmaterials that naturally luminesce or are predisposed to electrostaticbuild-up, either of which can increase the chances of a false positiveor interfering with quantification measurements.

The above-described process is repeated for each reaction tube 162 ofthe MTU 160. After the chemiluminescent signal from each reaction tube162 of the MTU 160 has been measured, the motor 972 advances to move theMTU 160 through the exit door 961 and out of the luminometer 950 andinto the amplicon deactivation station 750.

An alternate, and presently preferred, luminometer is generallydesignated by reference number 1360 in FIG. 43. Luminometer 1360includes a housing 1372 having a bottom wall 1370, door assemblies 1200on opposite sides of the bottom wall 1370 which define end portions ofthe housing 1372, an optical sensor shutter assembly 1250 which definesa front wall of the housing 1370, a top wall (not shown), and a backwall (not shown), which complete the housing 1370 and define anenclosure therein. The right-side door assembly 1200 defines areceptacle entrance opening 1374, and the left-side door assembly 1200defines a receptacle exit opening 1376 through which a MTU 160 can bepassed into and out of the housing 1370. Each door assembly 1200controls access through the respective opening 1374 or 1376 andcomprises an end wall 1202, a cover plate 1232, and a rotating door 1220rotatably disposed between the end wall 1202 and the cover plate 1232.The optical sensor aperture shutter assembly 1250 controls lightentering an optical sensor (not shown in FIG. 43), for example aphotomultiplier tube. Luminometer 1360 includes a light receivermounting wall 1250 and a cover plate 1290 having an aperture 1292 formedtherein.

A bar code scanner 1368 is attached to a front portion of the housing1372 for scanning MTUs prior to their entry to the luminometer 1360.

A receptacle transport assembly 1332 moves a receptacle (e.g., a MTU160) through the luminometer 1360 from the entrance opening 1374 to theexit opening 1376. The assembly 1332 includes a transport 1342 movablycarried on a threaded lead screw 1340 that is rotated by a motor 1336coupled to the lead screw 1340 by a belt (not shown).

A dispensing nozzle 1362 is attached in the top wall (not shown) and isconnected by conduit tubes 1364 and 1366 to a pump and ultimately tobottles 1146 and 1170 in the lower chassis 1100. Nozzle 1362 dispensesthe “Detect I” and the “Detect II” reagents into the receptacles 162 ofthe MTU 160 within the housing 1372.

A reaction tube positioner assembly 1300 is disposed within the housing1372 and is constructed and arranged to position each reaction tube 162of the MTU 160 in front of the aperture 1292 and to optically isolateeach reaction tube being positioned from adjacent reaction tubes, sothat only light from one reaction tube at a time enters the aperture1292. The positioner assembly 1300 comprises a receptacle positioner1304 rotatably mounted within a positioner frame 1302 that is secured tothe floor 1370 of the housing 1372.

The door assembly 1200 for the MTU entrance opening 1374 and exitopening 1376 of the luminometer 1360 is shown in FIG. 44. Door assembly1200 includes a luminometer end-wall 1202 which forms an end wall of theluminometer housing 1372. End-wall 1202 includes a first recessed area1206 with a second, circular recessed area 1208 superimposed on thefirst recessed area 1206. A circular groove 1207 extends about theperiphery of the circular recessed area 1208. A slot 1204, having ashape generally conforming to a longitudinal profile of an MTU 160, isformed in the circular recessed area 1208 to one side of the centerthereof. A short center post 1209 extends from the center of thecircular recessed area 1208.

The rotating door 1220 is circular in shape and includes an axial wall1222 extending about the periphery of the rotating door 1220. The axialwall 1222 is disposed a short radial distance from the outer peripheraledge of the rotating door 1220, thus defining an annular shoulder 1230about the outermost peripheral edge outside the axial wall 1222. A slot1226, having a shape generally conforming to the longitudinal profile ofan MTU is formed in the rotating door 1220 at an off-center position.

The rotating door 1220 is installed into the circular recessed area 1208of the end-wall 1202. A central aperture 1224 receives the center post1209 of the end-wall 1202, and circular groove 1207 receives axial wall1222. The annular shoulder 1230 rests on the flat surface of therecessed area 1206 surrounding the circular recessed area 1208.

End-wall 1202 includes a drive gear recess 1210 which receives therein adrive gear 1212 attached to the drive shaft of a motor 1213 (See FIG. 43in which only the motor 1213 for the right-side door assembly 1200 isshown). Motor 1213 is preferably a DC gear motor. A preferred DC gearmotor is available from Micro Mo Electronics, Inc. of Clearwater, Fla.,under Model No. 1524TO24SR 16/7 66:1. The outer circumference of theaxial wall 1222 of the rotating door 1220 has gear teeth formed thereonwhich mesh with the drive gear 1212 when the shutter is installed intothe circular recess 1208.

The cover plate 1232 is generally rectangular in shape and includes araised area 1234 having a size and shape generally conforming to therecessed area 1206 of the end-wall 1202. Cover plate 1232 has formedtherein an opening 1236 having a shape generally conforming to thelongitudinal profile of an MTU, and, when the cover plate 1232 isinstalled onto the end-wall 1202, the raised rectangular area 1234 isreceived within the rectangular recessed area 1206 and opening 1236 isin general alignment with opening 1204. Thus, the rotating door 1220 issandwiched between the cover plate 1232 and the end-wall 1202, and theopenings 1236 and 1204 together define the entrance opening 1374 andexit opening 1376.

When the drive gear 1212 is rotated by the motor 1213, the rotating door1220, enmeshed with the drive gear 1212, is caused to rotate about thecenter post 1209. When the opening 1226 is aligned with openings 1204and 1236, MTUs 160 can be passed through the opening 1374 (1376) of thedoor assembly 1200. With the rotating door 1220 disposed within thecircular recessed area 1208 and the raised area 1234 of the cover plate1232 disposed within the recessed area 1206 of the end-wall 1202, asubstantially light-tight structure is achieved, whereby little or nolight enters through the door, when the opening 1226 is not aligned withopenings 1204 and 1236.

Optical slotted sensors are disposed within slots 1214 and 1216 disposedon the outer edge of the circular recessed area 1208 at diametricallyopposed positions. Preferred sensors are available from OptekTechnology, Inc. of Carrollton, Tex., Model No. OPB857. The slottedsensors disposed within slots 1214 and 1216 detect the presence of anotch 1228 formed in the axial wall 1222 to signal door open and doorclosed status.

The optical sensor aperture shutter assembly 1250 is shown in FIG. 45. Alight receiver, such as a photomultiplier tube 956, is coupled with alight receiver opening 1254 formed in a light receiver mounting wall1252. The light receiver mounting wall 1252 includes a generallyrectangular, two-tiered raised area 1256, which defines a generallyrectangular shoulder 1257 and a circular recessed area 1258 superimposedon the rectangular raised area 1256. A circular groove 1261 extendsabout the periphery of circular recessed area 1258. A center post 1259is positioned at the center of the circular recessed area 1258. Lightreceiver opening 1254 is formed in the circular recessed area 1258. Inthe illustrated embodiment, the light receiver opening 1254 is disposedbelow the center post 1259, but the light receiver opening 1254 could beplaced at any position within the circular recessed area 1258.

The aperture shutter assembly 1250 includes a rotating shutter 1270having an axial wall 1274 with gear teeth formed on the outer peripherythereof. Axial wall 1274 is formed near, but not at, the outer peripheryof the shutter 1270, thereby defining annular shoulder 1276. Rotatingshutter 1270 is installed in the circular recessed area 1258 with centerpost 1259 received within a central aperture 1272 formed in the rotatingshutter 1270 and with axial wall 1274 received within circular groove1261. A drive gear 1262 disposed within a gear recess 1260 and coupledto a drive motor 1263 meshes with the outer gear teeth formed on theaxial wall 1274 of the rotating shutter 1270 to rotate the rotatingshutter 1270 about the center post 1259. A preferred drive motor 1263 isa DC gear motor available from Micro Mo Electronics, Inc. of Clearwater,Fla., as Model No. 1524TO24SR 16/7 66:1. Micro Mo gear motors arepreferred because they provide a high quality, low backlash motor. Anopening 1280 is formed in the rotating shutter 1270 which can be movedinto and out of alignment with light receiver opening 1254 as therotating shutter 1270 is rotated.

With the shutter 1270 installed in the circular recessed area 1258, acover plate, or sensor aperture wall, 1290 is installed onto the sensormount 1252. As shown in FIG. 45A, sensor aperture wall 1290 includes agenerally rectangular, two-tiered recessed area 1296 which defines agenerally rectangular shoulder 1297 and which is sized and shaped toreceive therein the rectangular raised area 1256 of the sensor mount1252. A sensor aperture 1292 is formed through the aperture wall 1290and is generally aligned with the light receiver opening 1254 formed inthe sensor mount 1252. The sensor aperture 1292 is generally in theshape of an elongated oval having a width generally corresponding to thewidth of an individual reaction tube 162 of an MTU 160 and a heightcorresponding to the height of the intended viewing area. Althoughopening 1280 of shutter 1270 is shown in the illustrated embodiment tobe circular, opening 1280 can have other shapes, such as rectangular,with a width corresponding to the width of a reaction tube 162 or anelongated oval similar to sensor aperture 1292. Rotation of the rotatingshutter 1270 to a position in which the opening 1280 is aligned with thelight receiver opening 1254 and the sensor aperture 1292 permits lightto reach the PMT 956, and rotation of the rotating shutter 1270 to aposition in which the opening 1280 is not aligned with light receiveropening 1254 and sensor aperture 1292 prevents light from reaching thePMT 956.

Slotted optical sensors are disposed in slots 1264 and 1266 and detect anotch 1278 formed in the axial wall 1274 of the shutter 1270 to detectopened and closed positions of the shutter 1270. Preferred slottedoptical sensors are available from Optek Technology, Inc., ofCarrollton, Tex., as Model No. OPB857.

The aperture wall 1290 includes an upwardly facing shoulder 1294extending across the width thereof. A downwardly facing shoulder of theMTU 160, defined by the connecting rib structure 164 of the MTU 160 (seeFIG. 58), is supported by the shoulder 1294 as the MTU 160 slidesthrough the luminometer.

The reaction tube positioner assembly 1300 is shown in FIGS. 46 and48-49. The reaction tube positioner 1304 is operatively disposed withinthe reaction tube positioner frame 1302. The reaction tube positioner1304 is mounted in the reaction tube positioner frame 1302 for rotationabout a shaft 1308. Shaft 1308 is operatively coupled to a rotarysolenoid, or, more preferably, a gear motor 1306, to selectively rotatethe reaction tube positioner 1304 between the retracted position shownin FIG. 46 and the fully extended position shown in FIG. 48. A preferredgear motor drive is available from Micro Mo Electronics, Inc. ofClearwater, Fla., as Model No. 1724T024S+16/7 134:1+X0520.

As shown in FIG. 47, the reaction tube positioner 1304 includes aV-block structure 1310 defining two parallel walls 1312. Reaction tubepositioner 1304 further includes an area at the lower end thereof wherea portion of the thickness of the reaction tube positioner 1304 isremoved, thus defining a relatively thin arcuate flange 1314.

When an MTU 160 is inserted into the luminometer 1360, the reaction tubepositioner 1304 is in the retracted position shown in FIG. 46. When anindividual reaction tube 162 is disposed in front of the sensor aperture1292 (see FIG. 45A), so that a sensor reading of the chemiluminescenceof the contents of the reaction tube 162 can be taken, the reaction tubepositioner 1304 rotates forwardly to the engaged position shown in FIG.49. In the engaged position shown in FIG. 49, the V-block 1310 engagesthe reaction tube 162, thus holding the reaction tube in the properposition in alignment with the light receiver aperture 1292 of theluminometer. As shown in FIG. 45, aperture wall 1290 includes aprotrusion 1298 extending from the back of wall 1290 into the MTUpassage of the luminometer. The protrusion 1298 is aligned with theaperture 1292 so that when the reaction tube positioner 1304 engages areaction tube 162, the reaction tube is pushed laterally and encountersprotrusion 1298 as a hard stop, thus preventing the reaction tubepositioner 1304 from significantly tilting the reaction tube 162 withinthe MTU passage. The parallel sidewalls 1312 of the V-block 1310 preventstray light from adjacent reaction tubes 162 of the MTU 160 fromreaching the light receiver while a reading is being taken of thereaction tube 162 disposed directly in front of the aperture 1292.

A slotted optical sensor 1318 is mounted to a lower portion of the frame1302, with the arcuate flange 1314 operatively positioned with respectto the sensor 1318. A preferred slotted optical sensor is available fromOptek Technology, Inc., of Carrollton, Tex., as Model No. OPB930W51. Anopening 1316 is formed in the flange 1314. Opening 1316 is properlyaligned with the sensor 1318 when the reaction tube positioner 1304engages a reaction tube 162 and the reaction tube 162 and protrusion1298 prevent further rotation of the reaction tube positioner 1304. If areaction tube 162 is not properly positioned in front of the reactiontube positioner 1304, the reaction tube positioner 1304 will rotateforwardly to the position shown at FIG. 48, in which case opening 1316will not be aligned with the sensor 1318 and an error signal will begenerated.

If a gear motor 1306 is employed for rotating the reaction tubepositioner 1304, it is necessary to provide a second sensor (not shown)to generate a positioner-retracted, i.e., “home”, signal to shut off thegear motor when the reaction tube positioner 1304 is fully retracted, asshown in FIG. 46. A preferred sensor is available from Optek Technology,Inc. of Carrollton, Tex. as Model No. OPB900W.

The MTU transport assembly 1332 is shown in FIG. 50. The MTU transportassembly 1332 is operatively positioned adjacent a top edge of anintermediate wall 1330 (not shown in FIG. 43) of the luminometer 1360.Intermediate wall 1330, which defines one side of the MTU transport paththrough the luminometer housing 1372, includes a rectangular opening1334. The reaction tube positioner frame 1302 (see, e.g., FIG. 48) ismounted to the intermediate wall 1330 proximate the opening 1334, andthe reaction tube positioner 1304 rotates into engagement with an MTU160 through the opening 1334.

The MTU transport 1342 is carried on the threaded lead screw 1340 andincludes a screw follower 1344 having threads which mesh with thethreads of the lead screw 1340 and an MTU yoke 1346 formed integrallywith the screw follower 1344. As shown in FIG. 51, the MTU yoke 1346includes a longitudinally-extending portion 1356 and twolaterally-extending arms 1348 and 1350, with a longitudinal extension1352 extending from the arm 1350. The lead screw 1340 is driven, via adrive belt 1338, by the stepper motor 1336. A preferred stepper motor isa VEXTA motor, available from Oriental Motors Ltd. of Tokyo, Japan,Model No. PK266-01A, and a preferred drive belt is available from SDP/SIof New Hyde Park, N.Y.

When an MTU 160 is inserted into the MTU transport path of theluminometer 950 by the right-side transport mechanism 500, the firstreaction tube 162 of the MTU 160 is preferably disposed directly infront of the sensor aperture 1292 and is thus properly positioned forthe first reading. The width of the yoke 1346 between the lateral arms1348 and 1350 corresponds to the length of a single MTU 160. Thetransport 1342 is moved between a first position shown in phantom inFIG. 50 and a second position by rotation of the lead screw 1340.Slotted optical sensors 1341 and 1343 respectively indicate that thetransport 1342 is in the either the first or second position. Due tofriction between the lead screw 1340 and the screw follower 1344, theMTU transport 1342 will have a tendency to rotate with the lead screw1340. Rotation of the MTU transport 1342 with the lead screw 1340 ispreferably limited, however, to 12 degrees by engagement of a lowerportion of the yoke 1346 with the top of the intermediate wall 1330 andengagement of an upper stop 1354 with the top cover (not shown) of theluminometer housing 1372.

To engage the MTU that has been inserted into the luminometer 1360, thelead screw 1340 rotates in a first direction, and friction within thethreads of the screw follower 1344 and the lead screw 1340 causes thetransport 1342 to rotate with lead screw 1340 upwardly until the upperstop 1354 encounters the top cover (not shown) of the luminometer 1360.At that point, continued rotation of the lead screw 1340 causes thetransport 1342 to move backward to the position shown in phantom in FIG.50. The lateral arms 1348, 1350 pass over the top of the MTU as thetransport 1342 moves backward. Reverse rotation of the lead screw 1340first causes the transport 1342 to rotate downwardly with the lead screw1340 until a bottom portion of the yoke 1346 encounters the top edge ofthe wall 1330, at which point the lateral arms 1348 and 1350 of the yoke1346 straddle the MTU 160 disposed within the luminometer 1360.

The MTU transport mechanism 1332 is then used to incrementally move theMTU 160 forward to position each of the individual reaction tubes 162 ofthe MTU 160 in front of the optical sensor aperture 1292. After the lastreaction tube 162 has been measured by the light receiver within theluminometer, the transport 1342 moves the MTU 160 to a position adjacentthe exit door, at which point the lead screw 1340 reverses direction,thus retracting the transport 1342 back, as described above, to aninitial position, now behind the MTU 160. Rotation of the lead screw1340 is again reversed and the transport 1342 is then advanced, asdescribed above. The exit door assembly 1200 is opened and thelongitudinal extension 1352 of the yoke 1346 engages the MTUmanipulating structure 166 of the MTU 160 to push the MTU 160 out of theluminometer exit door and into the deactivation queue 750.

Deactivation Station

In the amplicon deactivation station 750, dedicated delivery lines (notshown) add a deactivating solution, such as buffered bleach, into thereaction tubes 162 of the MTU 160 to deactivate nucleic acids in theremaining fluid in the MTU 160. Examples of nucleic acid deactivatingsolutions are disclosed in, for example, Dattagupta et al., U.S. Pat.No. 5,612,200, and Nelson et al., U.S. Patent Application PublicationNo. US 2005-0202491 A1. The fluid contents of the reaction tubes areaspirated by tubular elements (not shown) connected to dedicatedaspiration lines and collected in a dedicated liquid waste container inthe lower chassis 1100. The tubular elements preferably have a length of4.7 inches and an inside diameter of 0.041 inches.

An MTU shuttle (not shown) moves the MTUs 160 incrementally (to theright in FIG. 3) with the delivery of each subsequent MTU 160 into thedeactivation station 750 from the luminometer 950. Before an MTU can bedelivered to the deactivation queue 750 by the luminometer 950, the MTUshuttle must be retracted to a home position, as sensed by astrategically positioned optical slot switch. After receiving an MTU 160from the luminometer, the shuttle moves the MTU 160 to a deactivationstation where the dedicated delivery lines connected to dedicatedinjectors dispense the deactivating solution into each reaction tube 162of the MTU 160. Previous MTUs in the deactivation queue, if any, will bepushed forward by the distance moved by the MTU shuttle. Sensors at thedeactivation station verify the presence of both the MTU and the MTUshuttle, thus preventing the occurrence of a deactivating fluidinjection into a non-existent MTU or double injection into the same MTU.

An aspiration station (not shown) includes five, mechanically coupledaspirator tubes mounted for vertical movement on an aspirator tube rackand coupled to an actuator for raising and lowering the aspirator tubes.The aspiration station is at the last position along the deactivationqueue before the MTUs are dropped through a hole in the datum plate 82and into the waste bin 1108. Each time an MTU moves into thedeactivation station, the aspirator tubes cycle up and down one time,whether an MTU is present in the aspiration station or not. If an MTU ispresent, the aspirator tubes aspirate the fluid contents from the MTU.When the next MTU is moved into the deactivation station by the MTUshuttle, the last-aspirated MTU is pushed off the end of thedeactivation queue and falls into the waste bin 1108.

Ideally, the analyzer 50 can run about 500 preferred assays in an 8 hourperiod, or about 1,000 preferred assays in a 12 hour period. Once theanalyzer 50 is set-up and initialized, it ordinarily requires little orno operator assistance or intervention. Each sample is handledidentically for a given assay, although the analyzer is capable ofsimultaneously performing multiple assay types in which different MTUsmay or may not be handled identically. Consequently, manual pipetting,incubation timing, temperature control, and other limitations associatedwith manually performing multiple assays are avoided, thereby increasingreliability, efficiency, and throughput. And because an operator'sexposure to samples is generally limited to the loading of samples,risks of possible infection are greatly reduced.

Real-Time Amplification Assays

Real-time amplification assays can be used to determine the presence andamount of a target nucleic acid in a sample which, by way of example, isderived from a pathogenic organism or virus. By determining the quantityof a target nucleic acid in a sample, a practitioner can approximate theamount or load of the organism or virus in the sample. In oneapplication, a real-time amplification assay may be used to screen bloodor blood products intended for transfusion for bloodborne pathogens,such as hepatitis C virus (HCV) and human immunodeficiency virus (HIV),or to monitor the efficacy of a therapeutic regimen in a patientinfected with a pathogenic organism or virus. Real-time amplificationassays may also be used for diagnostic purposes, as well as in geneexpression determinations. In a preferred application of the presentinvention discussed above, the presence of an organism or virus ofinterest is determined using a probe which, under the particularconditions of use, exhibits specificity in a sample for a target nucleicacid sequence derived from the organism or virus of interest (i.e.,contained within target nucleic acid obtained from the organism or virusor an amplification product thereof). To exhibit specificity, a probemust have a nucleotide base sequence which is substantiallycomplementary to the target or its complement such that, under selectiveassay conditions, the probe will detectably hybridize to the targetsequence or its complement but not to any non-target nucleic acids whichmay be present in the sample.

In addition to the “end-point” amplification assays described above,where the amount of amplification products containing the targetsequence or its complement is determined in a detection station, such asthe luminometer 950, at the conclusion of an amplification procedure,the present invention is also able to perform “real-time” amplificationassays, where the amount of amplification products containing the targetsequence or its complement is determined during an amplificationprocedure. In the real-time amplification assay, the concentration of atarget nucleic acid can be determined by making periodic determinationsof the amount of amplification product in the sample which contains thetarget sequence, or its complement, and calculating the rate at whichthe target sequence is being amplified. Preferably, the instrument canbe selectively used in an end-point or real-time detection mode orsimultaneously in both modes.

For real-time amplification assays, the probes are preferablyunimolecular, self-hybridizing probes having a pair of interactinglabels which interact to emit different signals, depending on whetherthe probes are in a self-hybridized state or hybridized to the targetsequence or its complement. See, e.g., Diamond et al., “DisplacementPolynucleotide Assay Method and Polynucleotide Complex ReagentTherefor,” U.S. Pat. No. 4,766,062; Tyagi et al., “Detectably LabeledDual Conformation Oligonucleotide Probes, Assays and Kits,” U.S. Pat.No. 5,925,517; Tyagi et al., “Nucleic Acid Detection Probes HavingNon-FRET Fluorescence Quenching and Kits and Assays Including SuchProbes,” U.S. Pat. No. 6,150,097; and Becker et al., “MolecularTorches,” U.S. Pat. No. 6,361,945. Other probes are contemplated for usein the present invention, including complementary, bimolecular probes,probes labeled with an intercalating dye and the use of intercalatingdyes to distinguish between single-stranded and double-stranded nucleicacids. See, e.g., Morrison, “Competitive Homogenous Assay,” U.S. Pat.No. 5,928,862; Higuchi, “Homogenous Methods for Nucleic AcidAmplification and Detection,” U.S. Pat. No. 5,994,056; and Yokoyama etal., “Method for Assaying Nucleic Acid,” U.S. Pat. No. 6,541,205.Examples of interacting labels include enzyme/substrate,enzyme/cofactor, luminescent/quencher, luminescent/adduct, dye dimersand Förrester energy transfer pairs. Methods and materials for joininginteracting labels to probes for optimal signal differentiation aredescribed in the above-cited references.

In a preferred real-time amplification assay, the interacting labelsinclude a fluorescent moiety and a quencher moiety, such as, forexample, 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL). Thefluorescent moiety emits light energy (i.e., fluoresces) at a specificemission wavelength when excited by light energy at an appropriateexcitation wavelength. When the fluorescent moiety and the quenchermoiety are held in close proximity, light energy emitted by thefluorescent moiety is absorbed by the quencher moiety. But when a probehybridizes to nucleic acid present in the sample, the fluorescent andquencher moieties are separated from each other and light energy emittedby the fluorescent moiety can be detected. Fluorescent moieties whichare excited and emit at different and distinguishable wavelengths can becombined with different probes. The different probes can be added to asample, and the presence and amount of target nucleic acids associatedwith each probe can be determined by alternately exposing the sample tolight energy at different excitation wavelengths and measuring the lightemission from the sample at the different wavelengths corresponding tothe different fluorescent moieties.

In one example of a multiplex, real-time amplification assay, thefollowing may be added to a sample prior to initiating the amplificationreaction: a first probe having a quencher moiety and a first fluorescentdye (having an excitation wavelength λ_(ex1) and emission wavelengthλ_(em1)) joined to its 5′ and 3′ ends and having specificity for anucleic acid sequence derived from HCV; a second probe having a quenchermoiety and a second fluorescent dye (having an excitation wavelengthλ_(ex2) and emission wavelength λ_(em2)) joined to its 5′ and 3′ endsand having specificity for a nucleic acid sequence derived from HIV Type1 (HIV-1); and a third probe having a quencher moiety and a thirdfluorescent dye (having an excitation wavelength λ_(ex3) and emissionwavelength λ_(em3)) joined to its 5′ and 3′ ends and having specificityfor a nucleic acid sequence derived from West Nile virus (WNV). Aftercombining the probes in a sample with amplification reagents, thesamples can be periodically and alternately exposed to excitation lightat wavelengths λ_(ex1), λ_(ex2), and λ_(ex3), and then measured foremission light at wavelengths λ_(em1), λ_(em3), and λ_(em3), to detectthe presence (or absence) and amount of all three viruses in the singlesample. The components of an amplification reagent will depend on theassay to be performed, but will generally contain at least oneamplification oligonucleotide, such as a primer, a promoter-primer,and/or a promoter oligonucleotide, nucleoside triphosphates, andcofactors, such as magnesium ions, in a suitable buffer.

Where an amplification procedure is used to increase the amount oftarget sequence, or its complement, present in a sample before detectioncan occur, it is desirable to include a “control” to ensure thatamplification has taken place and, thereby, to avoid false negatives.Such a control can be a known nucleic acid sequence that is unrelated tothe sequence(s) of interest. A probe (i.e., a control probe) havingspecificity for the control sequence and having a unique fluorescent dye(i.e., the control dye) and quencher combination is added to the sample,along with one or more amplification reagents needed to amplify thecontrol sequence, as well as the target sequence(s). After exposing thesample to appropriate amplification conditions, the sample isalternately exposed to light energy at different excitation wavelengths(including the excitation wavelength for the control dye) and emissionlight is detected. Detection of emission light of a wavelengthcorresponding to the control dye confirms that the amplification wassuccessful (that is, that the control sequence was indeed amplified),and thus, any failure to detect emission light corresponding to theprobe(s) of the target sequence(s) is not likely due to a failedamplification. Conversely, failure to detect emission light from thecontrol dye is likely indicative of a failed amplification, thusrendering any results from that assay suspect.

Real-time amplification assays are performed in a real-time incubator(“RT incubator”), which is a modified version of the AT incubator 602described above. The RT incubator, designated by reference number 608 inFIGS. 61 and 64-65, is essentially a rotary incubator, such asincubators 600, 602, 604 and 606. The RT incubator includes instrumentsattached thereto for detecting, in a real-time manner, the amplificationoccurring within the reaction tubes 162 of an MTU 160 carried in the RTincubator by measuring the fluorescence emitted by a dye or dyes withineach reaction tube 162 of the MTU 160 when the MTU 160 is illuminatedwith an excitation light corresponding to each dye. The RT incubator 608can be integrated into the automated diagnostic analyzer 50 by modifyingthe AT incubator 602 so as to enable it to function as either the ATincubator 602 for end-point amplification assays or as the RT incubator608 for real-time amplification assays. Alternatively, the RT incubator608 can be secured on a structure (not shown) that is ancillary to thehousing 60 if it is desired to keep the AT incubator 602 separate fromthe RT incubator 608. In this case, it can be appreciated thatadditional mechanisms, such as transport mechanisms 500, 502 will benecessary to transport the MTU 160 from the processing deck 200 of theanalyzer 50 to the RT incubator 608 carried on an ancillary structureadjacent to the processing deck 200.

The instruments attached to the RT incubator 608 for real-timefluorescence detection are known as optical detection modules (a type ofa signal measuring device), as will now be described.

An optical detection module generally designated by reference number1700 is shown in side cross-section in FIG. 61. Also shown in FIG. 61 isa portion of the floor 613 of the RT incubator 608 with the opticaldetection module 1700 extending through an opening 615 formed in thefloor 613. A portion of the cylindrical wall 610 is shown, but, forclarity of illustration, insulating jacket 612 is not shown in FIG. 61.In addition, the datum plate 82, to which the RT incubator 608 ismounted and below which most of the optical detection module 1700 islocated, is not shown in FIG. 61. A portion of an MTU 160 is shownpositioned above the optical detection module 1700 with the opticaldetection module 1700 positioned below a first reaction tube 162 a ofthe MTU 160. Substantially identical optical detection modules 1700 arepreferably positioned with respect to each of the other reaction tubes162 b, 162 c, 162 d, and 162 e at different locations on the RTincubator 608.

As shown in FIGS. 61-63, the optical detection module 1700 includes ahousing 1710 attached to a printed circuit board 1790. The housing 1710includes four sections: the excitation light housing 1714, theexcitation lens housing 1712, the adaptor pipe 1718, and the emissionlens housing 1716. The excitation lens housing 1712, the excitationlight housing 1714, and the emission lens housing 1716 are eachpreferably formed from machined 6061-T6 aluminum with a black anodizefinish. The adaptor pipe 1718 is preferably formed from a Delrin® resin.As can be seen in FIG. 61, the adaptor pipe 1718 is in close proximityto the incubator floor 613 of the RT incubator 608. Accordingly, toprovide a level of thermal isolation of the optical detection module1700 from the RT incubator 608, the adaptor pipe 1718 is preferablyformed from a material having low thermal conductivity, such as aDelrin® resin. The adapter pipe 1718 also provides additional electricalisolation between the housing 1710 and the circuit board 1790.

The excitation light housing 1714 houses the excitation light assembly1730 (described in more detail below) and is attached at a lower endthereof to the printed circuit board 1790 and at an upper end thereof toan end of the excitation lens housing 1712. The excitation light housing1714 is attached to the excitation lens housing 1712 by means ofmechanical fasteners, such as screws (not shown). The assembly may alsoinclude location pins 1721 (see FIG. 62) extending between theexcitation light housing 1714 and the excitation lens housing 1712 tofacilitate precise relative positioning of the respective housingsduring assembly thereof.

The excitation lens housing 1712 includes a first portion 1713 orientedhorizontally in the illustration and a second portion 1715 extending ata right angle from the first portion 1713 and oriented vertically in thefigure. An oblique surface 1717 preferably has an angle of 45° withrespect to the longitudinal axes of the first portion 1713 and secondportion 1715.

The adaptor pipe 1718 includes a base portion 1720 adapted to mate in alight-tight manner with the end of the second portion 1715 of theexcitation lens housing 1712. More specifically, the base portion 1720of the adaptor pipe 1718 includes a projection 1724, preferably circularin shape (see FIG. 61), which extends into a recession of mating sizeand shape formed in the upper end of the second portion 1715 of theexcitation lens housing 1712. An upper portion 1722 of the adaptor pipe1718 projects above the base portion 1720. Upper portion 1722 ispreferably circular in shape and adapted to project through an opening615 formed in the floor 613. Upper portion 1722 preferably has a smallercross-wise dimension than that of the base portion 1720, thereby forminga shoulder 1726 between the upper portion 1722 and the base portion1720, the shoulder 1726 bearing against the bottom of the floor 613 whenthe optical detection module 1700 is installed on the RT incubator 608.

The adaptor pipe 1718 is preferably secured to the excitation lenshousing 1712 by means of mechanical fasteners, such as screws (notshown), and locator pins (not shown) may extend between the adaptor pipe1718 and the second portion 1715 of the excitation lens housing 1712 tofacilitate precise relative positioning of the respective parts duringthe assembly thereof.

The emission lens housing 1716 is attached at a lower end thereof to theprinted circuit board 1790 at a spaced apart position from theexcitation light housing 1714. An upper end of the emission lens housing1716 is in the form of an oblique surface 1719 having a preferred angleof 45° and conforming to the oblique surface 1717 of the excitation lenshousing 1712. The excitation lens housing 1712 and the emission lenshousing 1716 are preferably connected to one another by mechanicalfasteners such as screws (not shown) and may also include locating pins1723 extending between the housings to facilitate precise positioning ofthe housings during the assembly thereof.

Gasket material (not shown) may be placed on mating surfaces between anyof the respective housings to limit light infiltration into the housing1710. Such gasket material may, for example, comprise a foam material.

As shown in FIGS. 61 and 63, the internal optics of the opticaldetection module 1700 include an excitation light assembly 1730, anexcitation lens assembly 1740, and an emission lens assembly 1770.

The excitation light assembly 1730 includes a light emitting diode (LED)1732 connected in the conventional manner to the printed circuit board1790. Different fluorescent dyes are excited at different wavelengths.In one multiplex application of the present invention, preferred dyesinclude the rhodamine dyes tetramethyl-6-rhodamine (“TAMRA”) andtetrapropano-6-carboxyrhodamine (“ROX”) and the fluorescein dyes6-carboxyfluorescein (“FAM”) and2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyrhodamin (“JOE”), each incombination with a DABCYL quencher. The excitation spectra of thepreferred dyes are shown in FIG. 66. Because the preferred dyes areexcited at different wavelengths, the optical detection module 1700 ispreferably tailored to emit an excitation light at or near the desiredexcitation wavelength (i.e., color) for the particular dye for which theoptical detection module is intended. Accordingly, component selectionfor the optical system will in many instances be governed by theparticular dye for which the optical detection module is intended. Forexample, with respect to the LED 1732, the particular LED (manufacturerand model number) selection will depend on the dye for which the opticaldetection module is intended. For the FAM dye, the preferred LED isavailable from Kingbright Corporation, City of Industry, Calif., asModel No. L7113PBCH; for the TAMRA dye, the preferred LED is availablefrom Kingbright as Model No. L7113VGC/H; for the ROX dye, the preferredLED is available from Agilent Technologies, Inc., Palo Alto, Calif., asModel No. HLMP-EL16-VY000; and for the JOE dye, the preferred LED isavailable from Kingbright as Model No. L7113VGC/H.

A light pipe 1733 is positioned above the LED 1732 and includes a baseportion 1734 and an elongate portion 1735 projecting from the baseportion 1734. The light pipe 1733, also known as a mixing rod, ispreferably a molded or extruded transparent acrylic. The light pipe 1733takes light emitted by the LED 1732, transmits it upwardly away from theLED 1732 and creates a spatially homogenous light distribution at theend of the elongate portion 1735 of the light pipe 1733 opposite the LED1732. The light pipe acts as a light transmitter and a physical spacerwhich makes the height of the excitation light assembly 1730 conform tothe height of the emission lens assembly 1770. Also, spatiallyhomogeneous excitation light leads to better fluorometer reads andfacilitates radiometric repeatability. The light pipe 1733 extendsthrough a restricted opening 1711 formed in the interior of theexcitation light housing 1714.

Light from the light pipe 1733 is directed toward a mirror 1736 disposedat an upper end of the light pipe housing 1714. Preferred mirrorsinclude mirrors available from Edmund Optics Inc., Barrington, N.J., asPart No. Y43-790 (enhanced aluminum), and as Part No. Y43-791 (protectedgold). The mirror 1736 is preferably oriented at an angle of about 45°.A cover 1738, preferably made from a Delrin® resin or other suitablematerial, is attached to the upper end of the excitation light housing1714 above the mirror 1736. The mirror 1736 is installed into a counterbored opening formed in the upper end of the light pipe housing 1714,and thereafter the housing is closed by means of the cover 1738.

The excitation lens assembly 1740 includes a first lens 1744 and asecond lens 1746. Preferred lenses for the first lens 1744 includelenses available from Edmund Optics as Part No. Y32-913, and suitablelenses for the second lens 1746 include lenses available from EdmundOptics as Part No. Y45-348. Lenses 1746 and 1744 are separated from oneanother by a spacer element 1743, preferably formed from 6061-T6aluminum with a black anodize finish. Light reflected off the mirror1736 is directed through the lenses 1744 and 1746, which collimate thereflected light to within a preferred tolerance of +/−10°. Light passingthrough the second lens 1746 passes thereafter through a baffle aperture1750. The baffle aperture 1750 is a ring with an inner surface that isangled at 35° with respect to the longitudinal axis of the first portion1713 of housing 1712 (i.e., the optical axis) and is preferably madefrom machined 6061-T6 aluminum with a black anodize finish. The purposeof the baffle 1750 is to block out stray light that is not within the+/−10° tolerance. The second lens 1746 is separated from the aperturebaffle 1750 by means of a spacer element 1748, preferably made from6061-T6 aluminum with a black anodize finish.

Following the aperture baffle 1750, the light passes through anexcitation filter 1752 to remove unwanted spectral components of theexcitation light. Again, the specific filter used will depend on theexcitation spectra of the dye for which the optical detection module1700 is intended. The preferred filters are available from ChromaTechnology Corp., Rockingham, Vt., as Part No. HQ480/43x for the dyeFAM, as Part No. HQ525/50x for the dye TAMRA, as Part No. HQ590/20x forthe dye ROX, and as Part No. HQ514/22x for the dye JOE. The elements ofthe excitation lens assembly 1750 are held in place within theexcitation lens housing 1712 by means of a retainer ring 1742. Suitableretainer rings are available from Thorlabs, Inc. of Newton, N.J., asPart No. SM18RR.

Following the excitation filter 1752, the light impinges upon dichroicbeam splitter 1754. The dichroic beam splitter 1754 is oriented at 45°and redirects the excitation light passing through excitation filter1752 by 90° toward lens 1756 disposed in the adaptor pipe 1718(described below). The specific beam splitter employed depends on thedye for which the optical detection module 1700 is intended. Preferredbeam splitters are as follows: for the FAM dye, suitable beam splittersare available from Chroma Technology as Part No. 511 LP; for the TAMRAdye, suitable beam splitters are available from Chroma Technology asPart No 560LP; for the ROX dye, suitable beam splitters are availablefrom Chroma Technology as Part No. 615LP; and for the JOE dye, suitablebeam splitters are available from Chroma Technology as Part No 535LP.Lens 1756 is a focusing lens that directs light through window 1758,which then impinges upon a reaction tube 162 of the MTU 160. Suitablefocusing lenses are available from Edmund Optics as Part No. Y32-913.

When excited by the light of the correct bandwidth, and assuming thepresence of a particular dye in question, the contents of the reactiontube 162 will fluoresce, thereby emitting light. Light emitted by thecontents of a reaction tube 162 passes through the window 1758 and backthrough the lens 1756, which collects and focuses as much of the emittedlight as possible. The lens 1756 also collimates the emitted lightpreferably to within a tolerance of +/−10. The light passing through thelens 1756 thereafter impinges upon the dichroic beam splitter 1754 and,being of a different wavelength than the excitation light, the emittedlight passes through and is not redirected by the beam splitter 1754.After light passes through the dichroic beam splitter 1754, it passesinto the emission lens assembly 1770, where it first encounters anaperture baffle 1772. Baffle 1772 is preferably formed from 6061-T6aluminum with a black anodize finish and having an interior opening thatis angled at 35° with respect to its longitudinal axis. Aperture baffle1772 blocks light that is not within the +/−10° tolerance.

After passing through the aperture baffle 1772, the light encountersemission filter 1774, which removes unwanted spectral components presentin the emission light. The specific filter preferred depends on thewavelength of the light emitted from the specific dye for which theoptical detector module is intended. FIG. 67 shows the emission spectraof the preferred dyes used in association with the present invention.Preferred emission filters are as follows: for the FAM dye, suitablefilters are available from Chroma Technology as Part No. HQ533/24m; forthe TAMRA dye, suitable filters are available from Chroma Technology asPart No. HQ588/35m; for the ROX dye, suitable filters are available fromChroma Technology as Part No. HQ640/40m; and for the JOE dye, suitablefilters are available from Chroma Technology as Part No. HQ560/30m.

Light next passes through a focusing lens 1778. Suitable lenses areavailable from Edmund Optics as Part No. Y45-348. The emission light isfocused by the lens 1778 onto a photodiode 1780 which generates acurrent signal in proportion to the intensity of the emission light.Suitable photodiodes are available from UDT Sensors, Inc. of Hawthorne,Calif., as Model No. PIN-10DI. Lens 1778 and filter 1774 are separatedfrom one another by a spacer element 1776, preferably formed from6061-T6 aluminum with a black anodize finish. The elements of theemission lens assembly 1770, other than the photodiode 1780, are held inplace within the emission lens housing 1716 by means of a retainer ring1777. Suitable retainer rings are available from Thorlabs, Inc. ofNewton, N.J., as Part No. SM18RR. The photodiode 1780 is connected tothe printed circuit board 1790.

FIGS. 68A-68F illustrate a suitable circuit (including an amplifiercircuit that produces a voltage that is proportional to the currentgenerated by photodiode 1780) for circuit board 1790 which includes LED1732 and photodiode 1780.

The electronic circuit 1790 includes the following components andsub-circuits: power supply 1800 and power filters formed by capacitorsC6, C10, and C17 and resistors R11 and R25 (FIG. 68E), an excitationsource (LED) 1732 (FIG. 68F), excitation drive source circuit includingU1 and various components (FIG. 68F), receiver (photodiode) 1780 andvarious components (FIG. 68A), pre-amplifier circuit U6 (pins 5-7), U7,and various components (FIG. 68A), offset compensation circuit U6 (pins1-3) and various components (FIG. 68A), microprocessor circuit U5 andvarious components (FIG. 68B), analog switch circuit SW1 and variouscomponents (FIG. 68B), and low-pass differential filter circuit U2 andU3 (FIG. 68C), U4 (FIG. 68D) and various components.

In order to reject the effects of varying background ambient light,circuit 1790 incorporates microprocessor U5, which controls LED 1732(on/off) and creates a clock (set at 250 Hz for FAM/ROX, 350 Hz for TAM)that is used to modulate LED 1732 and control the analog switch SW1. Bymodulating the LED 1732 (excitation) and changing the state of theanalog switch SW1 (changing the gain of the subsequent differentialfilter U2 from positive to negative gain and back) at the samefrequency, a matched transmitter/receiver pair is created. Only thoseoptical signals arriving at the same frequency as this clock will beamplified; all ambient light and light signals modulated at a differentfrequency are suppressed.

A pre-amplifier (transimpedance) circuit—including U6 (pins 5-7) andU7—receives an electrical current from the photodiode 1780 and convertsit to an amplified voltage. In addition, the offset compensationcircuit—including U6 (pins 1-3)—provides a bias current that compensatesfor electrical current out of the photodiode 1780 that is in response toany ambient light (not modulated) incident on the photodiode 1780. Thisis so that ambient light (which can be many orders of magnitude greaterthan the modulated light of interest) does not saturate the output ofthe pre-amplifier which, given the gain in this pre-amplifier (20mV/nA), is easily and frequently accomplished.

Due to the high gain and the small signal being measured, thepre-amplifier circuit can be highly susceptible to errors in measurementas a result of EMI/RFI interference and changes in temperature andhumidity. To minimize these effects, circuit traces and componentscomprising high impedance circuits, especially the photodiode 1780 andconnected points, are located as far as possible from other circuits.Additionally, the printed circuit board 1790 is preferably constructedto facilitate the complete removal of contaminants that may collectadjacent critical high impedance components, especially components R33,R36 and C21. To minimize the amount of contaminants and residual fluxremaining on the circuit board 1790 after assembly, the board is firstwashed with saponifiers appropriate for the solder/flux to be used forsoldering and then rinsed with deionized water. Following thesepreparatory steps, the photodiode 1780 is preferably soldered to thecircuit board 1790 with a “no-wash flux” core solder and any residualflux remaining on the circuit board 1790 provides a protective barrierand, therefore, is preferably not removed. These steps should alleviatethe effects of long term drift and circuit sensitivity associated withchanges in temperature and humidity. In addition, the pre-amplifierportion of the circuit board 1790 is fully contained within a groundedhousing (Faraday cage) to suppress any EMI/RFI interference.

Referring to FIG. 68A, amplifiers U7 and U6 (pins 5-7) form the firsttwo stages of amplification of the optical signal. Components C20, C22,C24, C26, C27, C28, R35, and R45 provide power supplybypassing/filtering to the amplifiers. C18, D2, R32, and R34 form afiltered −2.5V power supply that biases the anode of the photodiode1780. Feedback resistors R33 and R36 convert electrical current from thephotodiode 1780 into a voltage while C21 provides filtering for signalsof frequency 3.6 KHz and higher. The voltage divider formed by R37 andR38 provide a voltage gain of 10 in the next pre-amplification stagewhile capacitor C23 provides additional low pass filtering.

Amplifier U6 (pins 1-3) creates a DC bias current that negates theelectrical current from the photodiode 1780 that is attributed tobackground ambient light and other natural DC offsets in the circuit.The circuit forms an integrating amplifier that generates an electricalcurrent that is fed back into the input of the initial pre-amplifiercircuit (formed by U7). This results in an output signal at U7 and atU6, pin 7 that has a zero DC component, i.e., the signal is centeredaround 0V.

Microprocessor U5 (FIG. 68B) controls LED function (e.g., turns off theLED 1732 or modulates the LED 1732 at its intended operating frequency,250 Hz for FAM and ROX, 350 Hz for TAM) and differential amplifier gain.Depending on its input signals (pins 6 and 7), microprocessor U5controls the LED 1732 into an ‘off’ or ‘modulated’ state. The gain ofthe differential filter circuit U2, U3, U4 is adjustable within therange of plus or minus twelve depending on the phase relationshipbetween the control signals to the LED 1732 and the analog switch SW1.

Referring to FIG. 68F, components C1, R3, and VR1 form a referencevoltage circuit, and these components, along with resistors R18 and R27,establish the LED current (when LED 1732 is turned on). Components R1,R2, and Q1 form the LED control circuit which, when the FET switch Q1 isturned on, the voltage at the input pin of the amplifier U1 is raised,forcing the amplifier's output to go to a low voltage, effectivelyturning off LED current switch Q2. If Q1 is not turned on, then U1controls the voltage on the gate of the FET switch Q2 such thatelectrical current through the LED 1732 is controlled at the establishedset-point. LED modulation frequency and off/on control is controlled bythe input ‘LED_ON’ from the microprocessor U5 described above (FIG.68B).

Referring to FIG. 68B, the analog switch SW1 is used to ‘invert’ theinput voltage to the differential filter that follows. The frequency atwhich the signal is inverted is set and controlled by the microprocessorU5. Depending on the setting of the inputs A0 and A1 of analog switchSW1, one set of switches (internal to the analog switch SW1) are turnedon, passing the signal as wired through the device (either switches S1Aand S2A are “on” or switches SIB and S2B are “on,” connected through tooutputs D1 and D2, respectively). In this circuit, the inputs to theanalog switch SW1 are wired such that when the LED 1732 is turned on,the signal out of the pre-amplifier U6 (pins 5-7) is directed to thepositive input of the differential filter U2 (pin 3) while ground isapplied to the negative input of the differential filter U2 (pin 5). Theoutput signal (after filtering) of the differential filter (U4) isroughly twelve times that amplitude. When the LED 1732 is turned off(while modulating), the output of the pre-amplifier circuit goesnegative and with approximately the same amplitude as when the LED 1732was turned on. The inputs to the differential filter now are wired theother way around, such that the negative signal out of the pre-amplifierU6 (pin 7) is directed to the negative input of the differential filterU2 (pin 5) while ground is applied to the positive input U2 (pin 3).Output signal (after filtering) is still approximately the sameamplitude as with the LED on and the analog switch in the otherposition.

The differential amplifier/filter U2, U3, U4 provides minimal gain (12×)and provides multi-pole low pass filtering of the signal (cutoff at 10Hz) while handling the signal differentially. This filter is used toattenuate any and all signals from the pre-amplifier that fall outsideof a 10 Hz range around the operating frequency of the LED/analogswitch, (240-260 Hz for FAM/ROX, 340-360 Hz for TAM). Attenuation of theelectrical signal conducted by the photodiode 1780 increases rapidly asfrequency deviates outside this range.

A final amplifier circuit (U4) functions as a difference amplifier withzero gain. Its function is to convert the voltage differential betweenthe two signals out of the differential filter into a positive voltagereferenced to circuit ground.

FIGS. 64 and 65 both show a top view of an RT incubator 608 andillustrate the positioning of optical detection modules 1700 mounted tothe bottom of the RT incubator 608. In the embodiment illustrated inFIGS. 64 and 65, the RT incubator includes 15 optical detection modules1700, 5 optical detection modules 1700 (one for each of the reactiontubes 162 a-162 e of the MTU 160) for each of three different dyes,namely FAM, TAMRA and ROX. Thus, on each of five radii corresponding toeach of the five reaction tubes 162 a-162 e of the MTU 160, there arethree optical detection modules 1700, one for each of the dyes. Theoptical detection modules 1700 are positioned at 24° increments aroundthe RT incubator. During detection at one particular module 1700, it ispossible that stray light from an adjacent reaction tube that is beingexcited at the same time can affect the emission detected at the module1700. In addition, excitation light can scatter off a reaction tube 162and excite adjacent reaction tubes 162. This condition is known ascross-talk. The optical detection modules are preferably positioned soas to maximize the distance between the detection windows of adjacentoptical detection modules 1700, thereby minimizing cross-talk betweenadjacent optical detection modules 1700. Cross-talk can also beprevented by providing light isolating baffles (not shown) in the formof concentric circular walls positioned between adjacent reaction tubes162 of the MTU 160.

Another method for reducing cross-talk between the emissions of adjacentreaction tubes and for subtracting background signals due to, forexample, stray light, is by using phase-synchronous detectiontechniques. The excitation light is frequency modulated by applying asignal of known frequency to LED 1732. For the TAMRA dye, an excitationsignal frequency of 350 Hz is used, and for the FAM and ROX dyes, anexcitation signal frequency of 250 Hz is used. Accordingly, theresulting emission light will display a frequency that is governed bythe frequency of the excitation light, and any emission signal having afrequency that is inconsistent with the frequency of the excitationlight can be discarded as not resulting from the excitation light. Knownphase-detector circuits can be used to output a voltage that isproportional to the phase difference between the excitation and emissionsignals.

As shown in FIG. 65, optical detection modules 1700 are preferablygrouped according to the dye for which the module is intended. That is,modules 1-5 are intended for the FAM dye, modules 6-10 are intended forthe TAMRA dye, and modules 11-15 are intended for the ROX dye. It hasbeen discovered that cross-talk is actually worse between adjacentoptical detection modules with excitation signals of differentwavelengths than between adjacent optical detection modules withexcitation signals of the same wavelength. Thus, groupinglike-wavelength detectors together, as shown in FIG. 65, reducescross-talk.

Also, to minimize the transmissions and reflections of stray light, theinterior components of an RT incubator 608 are preferably black incolor.

In a preferred embodiment, the optical detection modules 1700 mounted tothe bottom floor 613 of the RT incubator 608, and disposed mostly belowthe datum plate 82 of the processing deck 200, are surrounded by ashield (known as Faraday shield, not shown) which blocks strayelectromagnetic interference which can affect the optical detectionmodules.

FIGS. 64 and 65 show an RT incubator 608 with 15 optical detectionmodules 1700. Such an arrangement permits real-time scanning for a5-reaction tube 162 MTU 160 and three dyes. If it becomes desirable toincorporate a fourth dye into the procedure, for example as describedabove for detecting amplification products associated with threedifferent viruses and an internal control, it would be necessary, inthis embodiment, to incorporate 20 optical detection modules into the RTincubator 608, which, as can be appreciated from FIGS. 2 62 and 63,would be nearly impossible given space constraints and the size of theillustrated embodiment of the optical detection module 1700.

To avoid the need for 20 optical detection modules, i.e., 5 opticaldetection modules for each of the four individual dyes, a scanningreal-time fluorometer can be used in which four optical detectionmodules, one for each dye, are mounted so as to be movable with respectto the RT incubator 608, so that each optical detection module can beselectively positioned beneath each of the five reaction tubes 162 a-162e of the MTU 160. (The number of optical detection modules can beadjusted in the scanning real-time fluorometer based on the number ofdyes to be detected.) A scanning fluorometer assembly for such ascanning real-time fluorometer is designated generally by referencenumber 2000 in FIGS. 69-72. In the scanning fluorometer assembly shown,four optical detection modules 1700 are mounted so as to be movable in aradial direction with respect to a fixed scanning disk 2002. Eachoptical detection module 1700 is mounted into an optical detectionmodule mounting bracket 2004 which is carried on a translating assembly2006 for effecting radial movement of the optical detection module 1700with respect to the scanning disk 2002. The translating assembly 2006comprising a sliding, linear bearing 2008 to which the mounting bracket2004 is attached and a bearing track 2010 mounted to the scanning disk2002 and along which the bearing 2008 is slidably translatable. Aslotted optical sensor 2014 is secured to the scanning disk 2002 at theradially outward end of the bearing track 2010, and a projection 2016extending from the bearing 2008 extends into the sensor 2014 when theoptical detection module 1700 is at the furthest out radial position,thereby providing a “home” signal. Generally radial slots 2040 areformed in the scanning disk 2002 (see FIG. 72).

FIG. 72 shows the bottom plan view of the scanning fluorometer assembly2000. A cam disk 2030 is arranged coaxially with and parallel to thescanning disk 2002 and is rotatable about shaft 2024 (see FIG. 71). Thecam disk 2030 has four arcuate cam slots 2032 formed therein, one foreach of the optical detection modules 1700. A pin 2050 extending downfrom the bearing 2008 of each translating assembly 2006 extends throughthe radial slot 2040 and into a respective one of the cam slots 2032.

A motor 2020 is mounted on top of the fixed scanning disk 2002. Anoutput shaft 2022 of the motor is coupled to the cam disk 2030 (e.g., bya belt and pulley arrangement (not shown) or a meshing gear arrangement(not shown)). A preferred embodiment employs a pulley arrangement with a6:1 ratio which achieves a speed of 50° in 0.25 seconds (i.e.,200°/sec.). Rotation of the output shaft 2022 coupled to the cam disk2030 causes rotation of the cam disk 2030. As the cam disk 2030 rotates,the engagement of each pin 2050 with a respective one of the cam slots2032 formed in the cam disk 2030 causes radial, translating motion of acorresponding one of the bearings 2008 along its respective bearingtrack 2010, thereby causing radial translation of the correspondingmodule mounting bracket 2004 and optical detection module 1700. Anencoder (not shown) on the motor monitors motor rotations, therebyallowing the position of the cam disc 2030 to be monitored.Alternatively, other position sensing devices, such as optical sensors,can be used to directly monitor the cam disk 2030 position.

Each cam slot 2032 includes positioning points 2032 a, 2032 b, 2032 c,2032 d, and 2032 e. (To minimize clutter, the positioning points 2032 a,2032 b, 2032 c, 2032 d, and 2032 e are labeled for only one of the camslots 2032.) The positioning points 2032 a-e position the opticaldetection modules 1700 during rotation of the cam disk 2030. When thepin 2050 associated with a particular optical detection module 1700 isat position 2032 a the optical detection module 1700 is at the radialposition furthest out from the center of disk 2002 for scanning thefurthest out reaction tube 162 a of the MTU 160. As the cam disk 2030rotates and the pin 2050 associated with the optical detection module1700 moves to point 2032 b, the corresponding optical detection modulewill move radially inwardly by a distance corresponding to the distanceto the next reaction tube 162 b of the MTU 160. The optical detectionmodule 1700 can then scan the reaction tube 162 b at that position.Further rotation of the cam disk 2030 causes the pin 2050 associatedwith the module 1700 to move to position 2032 c, thereby moving themodule 1700 radially inwardly by a distance corresponding to thedistance to reaction tube 162 c of the MTU 160. The module 1700 can thenscan reaction tube 162 c at that position. Further rotation of the camdisk 2030 causes the pin 2050 associated with the module 1700 totranslate radially inwardly by a distance corresponding to the distanceto reaction tube 162 d. The module 1700 can then scan reaction tube 162d at that position. Further rotation of the cam disk 2030 causes the pin2050 associated with the module 1700 to translate inwardly by a distancecorresponding to the distance to reaction tube 162 e. The module 1700can then scan the reaction tube 162 e at that position. Accordingly, asingle movable optical detection module 1700 can detect emissions fromeach of the reaction tubes 162 a-e of each MTU 160.

Each optical detection module 1700 of the scanning fluorometer assembly2000 extends up into the incubator housing of the RT incubator 608.Since the detectors 1700 move radially, elongated, radial openings (notshown) are formed in the floor 613 of the RT incubator 608 through whicheach optical detection module scans the MTUs 160 within the RTincubator. In one embodiment, a shutter mechanism (not shown) ispositioned in each radial opening. The shutter mechanism has a movableopening through which the adapter pipe 1718 of each optical detectionmodule 1700 extends. As the optical detection module 1700 translatesradially, the opening of the shutter mechanism translates with it whilethe rest of the radial opening remains closed, thereby limiting heatloss and stray light through the radial opening.

As an alternative to the optical detection module described above, theoptical detection module may be a multiple wavelength fluorometer, forexample, a fluorometric microscope with a filter changer or afluorometric microscope with multiple bandwidth filters and multiplebandwidth beam splitters.

The inventors have determined that the magnetic particles used fortarget capture in a preferred embodiment of the present invention canaffect real-time detection of amplification products. Two particularinterfering effects have been identified. First, magnetic particles caninhibit amplification by adsorption of oligonucleotides (e.g.,amplification oligonucleotides and probes) and enzyme reagents (e.g.,nucleic acid polymerases). In addition, the presence of magneticparticles (settled or in suspension) can result in the dissipation ofthe fluorescence, thereby blocking or partially blocking the amount ofexcitation light that reaches the detection dyes and the amount of lightemitted from the reaction tubes 162 of the MTUs 160. This is known asthe black cloud effect.

To minimize this effect, in one embodiment of the RT incubator 608 amagnetic divider 1500 is provided as shown in FIGS. 73, 74, 75, and 75A.In a preferred embodiment of the invention, the RT incubator 608 holds15 MTUs 160 at a time, each spaced at 24° increments around thecarousel. Assuming a 30-position carousel, such as carousel 1656, isused, this means that only every other MTU station 1663 holds an MTU 160in the RT incubator 608. Thus, as shown in the figures, the magneticdivider 1500 can be positioned so as to span every other MTU station1663 on the carousel 1656 (described above), thereby leaving only 15 of30 stations available to receive an MTU 160. In alternative embodiments,magnet holders could be constructed to fit between each of the 30stations or to be positioned adjacent every other station, therebypermitting the contents of every other MTU 160 in the RT incubator 608to be processed in accordance with an alternative assay procedure. Suchmagnet holders may be formed from a ferrous sheet metal to which themagnets will adhere. A ferrous sheet metal material would also have theadvantage of greatly reducing the magnetic field on the opposite side ofthe magnets.

As shown primarily in FIGS. 74 and 75A, which, for simplicity, show onlya single magnetic divider 1500, the magnetic divider includes a magnetholder 1502 which comprises a magnet block 1504 and an attachment arm1510. A rectangular recessed area 1506 is formed in the magnet block1504, and openings 1508 are formed in the magnet block 1504 to receivelike sized and shaped magnets 1520. In the illustrated embodiment, theopenings 1508 are circular and the magnets 1520 are disc shaped.Currently preferred magnets are nickel plate coated neodymium-iron-borondiscs measuring ½ inch (diameter) by ⅛ inch (thickness) and having a Brmax of 12,100 and a Bh max of MGOe (ForceField, Fort Collins, Colo.;Item No. 0022). The magnets 1520 are placed within the associatedopenings 1508 and are held within the block 1504 by means of a retainerplate 1522, which may be secured to the magnetic holder 1502 by means ofa mechanical fastener, such as a screw or a bolt (not shown), passingthrough openings 1524 and 1526 formed in the retainer plate 1522 and themagnet block 1504, respectively.

The attachment arm 1510 extends from the magnet block 1504 and includesfastener holes 1512 which align with corresponding fastener holes 1661formed in a lower plate 1662 and dividers 1660 of the carousel 1656. Themagnetic dividers 1500 can be secured to the carousel 1656 by means ofsuitable mechanical fasteners, such as screws or bolts (not shown),extending through the fastener holes 1512 and 1661.

The magnetic dividers 1500 may also include an inboard arm 1528 (seeFIG. 75). Inboard arm 1528 stabilizes the magnetic divider 1500 andprovides an additional attachment point for attaching the magneticdivider 1500 to the carousel 1656.

As shown in FIGS. 75 and 75A, when an MTU 160′ is placed into an MTUslot in the carousel 1656, each reaction tube 162′ is positionedadjacent to one of the magnets 1520 carried in the magnetic divider1500. The magnet 1520 will cause at least portion of the magneticparticles to be drawn toward the wall of the reaction tube 162′ adjacentthe magnet 1520, thereby leaving a substantially reduced concentrationof magnetic particles in suspension within the remainder of the contentsof the reaction tube 162′ or settled on the bottom of the reaction tube162′.

As noted above, the preferred material for the MTU 160 is polypropylene.Polypropylene has, however, been determined to autofluoresce undercertain conditions. Accordingly, alternative MTU materials, such asacrylics, polystyrene, and cyclic olefins are contemplated. Also, asillustrated in FIGS. 75 and 75A by reaction tubes 162′ of MTU 160′, toconcentrate the sample in the bottom of the individual reaction tubes162′ of the MTU 160—thereby facilitating more consistent excitation ofand emission from the sample and to permit use of smaller reagentvolumes—it is contemplated to use an MTU having reaction tubes withfrustoconically shaped ends, instead of the rounded ends of the reactiontubes 162 of the MTU 160 shown, for example, in FIG. 74.

The process steps of real-time and end-point amplification assaysperformed in accordance with the present invention are illustrated inthe flow chart shown in FIG. 76. (FIG. 76A shows the steps of a completereal-time TMA amplification assay and those of an end-point TMAamplification assay through amplification; FIG. 76B shows the steps ofthe end-point TMA amplification assay after exposing the contents of thereaction tubes 162 to amplification conditions.) The steps describedrepresent exemplary TMA procedures only. Persons of ordinary skill willrecognize that the steps described below may be varied or omitted orthat other steps may be added or substituted in accordance with otherreal-time and end-point amplification assay procedures now known or yetto be developed. Reagent formulations for performing a host ofamplification procedures are well known in the art and could be used inor readily adapted for use in the present invention. See, e.g., Kacianet al., U.S. Pat. No. 5,399,491; Becker et al., U.S. Patent ApplicationPublication No. US 2006-0046265 A1; Linnen et al., Compositions andMethods for Detecting West Nile Virus, U.S. Patent Application No. US2004-0259108 A1; Weisburg et al., “Compositions, Methods and Kits forDetermining the Presence of Trichomonas Vaginalis in a Test Sample,”U.S. Patent Application Publication No. US 2004-0235138 A1; and Linnenet al., “Compositions and Methods for Determining the Presence of SARSCoronavirus in a Sample,” U.S. patent application Ser. No. 10/825,757,which enjoys common ownership herewith.

The process steps of the exemplary real-time and end-point TMAamplification assays begin with step 1902, in which an MTU 160 is movedto a pipetting position in the sample transfer station 250 below thesample preparation opening 252 provided in the jig plate 130. In step1904, the sample pipette assembly 450 dispenses 400 μL of a targetcapture reagent (“TCR”) into each reaction tube 162 of the MTU 160. Thetarget capture reagent includes a capture probe, a detergent-containinglytic agent, such as lithium lauryl sulfate, for lysing cells andinhibiting the activity of RNAses present in the sample material, andabout 40 μg Sera-Mag™ MG-CM Carboxylate Modified (Seradyn, Inc.,Indianapolis, Ind.; Cat. No. 24152105-050250), 1 micron,super-paramagnetic particles having a covalently bound poly(dT)₁₄. Thecapture probe includes a 5′ target binding region and a 3′ region havinga poly(dA)₃₀ tail for binding to the poly(dT)₁₄ bound to the magneticparticle. The target binding region of the capture probe is designed tobind to a region of the target nucleic acid distinct from the regionstargeted by the primers and the detection probe.

In step 1906, the pipette assembly 450 dispenses 500 μL of sample intoeach of the reaction tubes. In step 1908, the right-side transportmechanism 500 moves the MTU 160 to the right-side orbital mixer 550 tomix the sample and the TCR, preferably at 10 Hz for 30 seconds. Notethat the times given in FIG. 76 and the description thereof are desiredtimes, and the actual times may, in practice, vary from the givendesired times.

In step 1910, the right-side transport mechanism 500 moves the MTU 160from the right-side orbital mixer 550 to one of the temperature rampingstations 700 located under the jig plate 130. The MTU 160 preferablyresides in the temperature ramping station 700 at a temperature of 65°C. for 312 seconds. In step 1912, the right-side transport mechanism 500moves the MTU 160 from the ramping station 700 to the TC incubator 600where it resides at 62° C. for 20 minutes for hybridization of thecapture probe to target nucleic acids which may have been extracted fromthe sample. (At this temperature, there will be no appreciablehybridization of the capture probe to the immobilized poly(dT)₁₄oligonucleotide.) In step 1914, the left-side transport mechanism 502moves the MTU 160 from the TC incubator to one of the temperatureramping stations 700 located on the left-side of the processing deck200, where it is held for 174 seconds at ambient temperature. In step1916, the left-side transport mechanism 502 moves the MTU 160 from theramping station 700 to the AMP incubator 604, where the MTU resides at43° C. for 838 seconds to allow for immobilized oligonucleotidesassociated with the magnetic particles to bind to the capture probes.

In step 1918, the left-side transport mechanism 502 moves the MTU 160from the AMP incubator 604 to the left-side orbital mixer 552. Theleft-side orbital mixer 552 includes dispensers for dispensing, amongother substances, oil into the MTU 160. In the left-side orbital mixer552, 200 μL of silicone oil, a surface treating agent, are added to eachreaction tube 162 of the MTU 160, and the MTU is mixed at 12 Hz for 30seconds. In step 1920, the left-side transport mechanism 502 moves theMTU 160 from the left-side orbital mixer 552 to one of the magneticseparation stations 800 for the magnetic separation wash proceduredescribed above.

An advantage of adding a surface treating agent, such as silicone oil,to the sample solution in step 1918 is that it reduces the amount ofmaterial that adheres to the inner surfaces of the reaction tubes 162during the rinsing and aspiration steps of a magnetic separation washprocedure, thereby facilitating a more effective magnetic separationwash procedure. Although the MTUs 160 are preferably made of ahydrophobic material, such as polypropylene, small droplets of material,such as wash solution, may still form on the inner surfaces of the MTUreaction tubes 162 during the aspiration steps of a magnetic separationwash procedure. If not adequately removed from the reaction tubes 162during the magnetic separation wash procedure, this residual material,which may contain nucleic acid amplification inhibitors, could affectassay results. In alternative approaches, the surface treating reagentcould be added to the reaction tubes 162 and removed prior to adding TCRand sample or the surface treating agent could be added to the reactiontubes after TCR and sample have been aspirated from the reaction tubes,possibly with the wash solution, and then removed prior to addingamplification and enzyme reagents to the reaction tubes. The objectiveis to provide inner surfaces of the reaction tubes 162 with a coating ofthe surface treating agent. Inhibitors of amplification reactions areknown in the art and depend on the sample source and amplificationprocedure to being used. Possible amplification inhibitors include thefollowing: hemoglobin from blood samples; hemoglobin, nitrates, crystalsand/or beta-human chorionic gonadotropin from urine samples; nucleases;proteases; anionic detergents such as sodium dodecyl sulfate (SDS) andlithium lauryl sulfate (LLS); and EDTA, which is an anticoagulant andfixative of some specimens that binds divalent cations like magnesium,which, as noted above, is a cofactor used in nucleic acid-basedamplification reactions. See, e.g., Mahony et al., J. Clin. Microbiol.,36(11):3122-2126 (1998); Al-Soud, J. Clin. Microbiol., 39(2):485-493(2001); and Kacian et al., “Method for Suppressing Inhibition ofEnzyme-Mediated Reactions By Ionic Detergents Using High Concentrationof Non-Ionic Detergent,” U.S. Pat. No. 5,846,701.

In step 1922, the left-side transport mechanism 502 moves the MTU 160from the magnetic separation station 800 back to the left-side orbitalmixer 552 and 200 μL of silicone oil are added to each reaction tube 162of the MTU 160 to prevent evaporation and splashing of the fluidcontents during subsequent manipulations. In step 1924, the reagentpipette assembly 470 dispenses 75 μL of an amplification reagent intoeach reaction tube 162 of the MTU 160 disposed within the left-sideorbital mixer 552. For the exemplary TMA reactions, the amplificationreagents contain an antisense promoter-primer having a 3′ target bindingregion and a 5′ promoter sequence recognized by an RNA polymerase, asense primer that binds to an extension product formed with thepromoter-primer, nucleoside triphosphates (i.e., dATP, dCTP, dGTP, dTTP,ATP, CTP, GTP and UTP), and cofactors sufficient to perform a TMAreaction. For the real-time TMA amplification assay, the amplificationreagent also contains a strand displacement, molecular torch probeshaving interacting label pairs (e.g., interacting fluorescent andquencher moieties joined to the 5′ and 3′ ends thereof by conventionalmeans) and a target specific region capable of detectably hybridizing toamplification products as the amplification is occurring and,preferably, not to any non-target nucleic acids which may be present inthe reaction tubes 162. See Kacian et al., U.S. Pat. No. 5,399,491;Becker et al., “Single-Primer Nucleic Acid Amplification,” U.S. PatentApplication Publication No. US 2006-0046265 A1 (discloses an alternativeTMA-based amplification assay in which an antisense primer and a sensepromoter oligonucleotide blocked at its 3′ end are employed to minimizeside-product formation); and Becker et al., U.S. Pat. No. 6,361,945. TheMTU 160 is then mixed for 15 seconds at 16 Hz.

In step 1926, the left-side transport mechanism 502 moves the MTU 160from the left-side orbital mixer 552 to one of the temperature rampingstations 700 located on the left-side of the processing deck 200. TheMTU 160 is then incubated at 65° C. for 132 seconds. In step 1928, theleft-side transport mechanism 502 moves the MTU 160 from the temperatureramping station 700 to the TC incubator 600, where it is incubated for10 minutes at 62° C. for binding of the promoter-primer to a targetnucleic acid. The preferred promoter-primer in this particular TMAexample has a promoter sequence recognized by a T7 RNA polymerase. Instep 1930, the left-side transport mechanism 502 moves the MTU 160 fromthe TC incubator 600 to the AMP incubator 604, where the MTU 160contents are incubated at 43° C. for 10 minutes to stabilize the MTUcontents.

In step 1932, the reagent pipette assembly 470 adds 25 μL of an enzymereagent held at 20° C. from the reagent cooling bay 900 to each reactiontube 162 of the MTU 160 located in the AMP incubator 604. (Bymaintaining the temperature of the contents of each reaction tube 162 ata temperature slightly higher than the amplification temperature, theheat-sensitive enzymes can be maintained at a cool temperature prior toinitiating amplification.) The enzyme reagent of this example contains areverse transcriptase and a T7 RNA polymerase for performing TMA, atranscription-based amplification procedure. In step 1934, the linearmixer 634 within the AMP incubator 604 mixes the MTU 160 to which theenzyme reagent has been added for 15 seconds at 10 Hz, and thetemperature of the contents of each reaction tube 162 drops to about 42°C. In step 1936 the left-side transport mechanism 502 moves the MTU 160from the AMP incubator 604 to the RT incubator 608. The MTU 160 ismaintained in the RT incubator 608 at 42° C. for 60 minutes to permitamplification of target sequences and, for real-time amplifications,readings are taken at the prescribed frequency to detect hybridizationof the probe to amplification product during the amplification process.Since MTUs 160 are being processed in a continual manner through theinstrument 50 (typically, a new MTU begins the assay process every 165seconds), MTUs are continually being added to and removed from(typically every 165 seconds) the RT incubator 608. In step 1938, afterthe last reading has been taken, the right-side transport mechanism 500moves the MTU 160 from the RT incubator 608 to the luminometer 1360. Instep 1940, the MTU 160 passes from the luminometer 1360 to thedeactivation queue 750. Once in the deactivation queue 750, 2 mL of ableach-based agent are provided to each of the reaction tubes 162 todeactivate nucleic acid (i.e., alter the nucleic acid such that it isnon-amplifiable) present in the reaction tubes. See, e.g., Dattagupta etal., U.S. Pat. No. 5,612,200, and Nelson et al., U.S. Patent ApplicationPublication No. US 2005-0202491A1.

Following step 1936, an MTU 160 having contents being processed inaccordance with the exemplary end-point TMA amplification assay proceedsas shown in FIG. 76B. In step 1942 of this process, the left-sidetransport mechanism 502 transfers the MTU 160 from the RT incubator 608to a temperature ramping station 700 on the left-side of the processingdeck 200, where it is heated at 64° C. for 362 seconds. Alternatively,the MTU 160 is moved from the RT incubator 608 to a designated region ofthe HYB incubator 606 for temperature ramping. In step 1944, theleft-side transport mechanism 502 moves the MTU 160 from the temperatureramping station 700 to the HYB incubator 606, where 100 μL of probereagent is added to each reaction tube 162. The probe reagent contains asufficient amount of a probe for detectably binding to an amplificationproduct of the target nucleic acid and, preferably, not to anynon-target nucleic acids which may be present in the reaction tubes 162.For the exemplary end-point TMA embodiment, the probe is synthesized toinclude a non-nucleotide linker which is used for labeling the probewith a chemiluminescent acridinium ester. See Arnold et al., U.S. Pat.Nos. 5,185,439 and 6,031,091. In step 1946, the MTU 160 is positionedwithin the HYB incubator 606 adjacent the skewed disk linear mixer 634,which is employed to mix the contents of the MTU for 15 seconds at 14Hz. In step 1948, the contents of the MTU 160 are incubated at 64° C.for 1762 seconds.

For detection, the contents of each reaction tube 162 of the MTU 160 arefirst provided with 250 μL of a selection reagent in step 1950. Asdiscussed above, the selection reagent in the HPA assay contains analkaline reagent that specifically hydrolyzes acridinium ester labelsassociated with unhybridized probe, while acridinium ester labelsassociated hybridized probe are not hydrolyzed under these conditionsand can chemiluminesce in a detectable manner under the conditionsdescribed below, thereby permitting the user to distinguish betweenbound probe and probe free in solution. See Arnold et al., U.S. Pat. No.5,639,604. After adding the selection reagent to the reaction tubes 162,the MTU 160 is positioned adjacent the skewed disk linear mixer 634 andthe contents of the reaction tubes are mixed for 30 seconds at 13 Hz. Instep 1954, the contents of the reactions tubes 162 are incubated for 606seconds at 64° C. to facilitate the selection process.

In step 1956, the left-side transport mechanism 502 transfers the MTU160 from the HYB incubator 606 to the AMP incubator 604 to cool thecontents of the reaction tubes 162 at 43° C. for 172 seconds. Droppingthe temperature of the contents of the reaction tubes 162 below 50° C.will generally arrest the activity of the selection reagent, thereforeit is important to cool the contents of the reaction tubes of each MTU160 at substantially the same rate so that the final signal values fromthe various reaction tubes are comparable. In step 1958, the left-sidetransport mechanism 502 moves the MTU 160 from the AMP incubator 604 tothe RT incubator 608, after which the right-side transport mechanism 500moves the MTU 160 from the RT incubator 608 to a parking station 210 onthe right-side of the processing deck 200, where the contents of thereaction tubes 162 are further cooled at ambient temperature for 560seconds. In step 1960, the MTU 160 is transferred to a temperatureramping station 700 on the right-side of the processing deck 200 to coolthe contents of the reaction tubes 162 at 21° C. for 366 seconds. In anHPA multiplex assay involving multiple chemiluminescent labels, it ispreferable to keep the temperature of the contents of the reaction tubes162 below 29° C. so that the light-off characteristics of the labels aredistinguishable. See, e.g., Nelson et al., “Compositions for theSimultaneous Detection and Quantitation of Multiple Specific NucleicAcid Sequences,” U.S. Pat. No. 5,756,709.

In step 1962, the right-side transport mechanism 500 moves the MTU 160from the ramping station 700 to the luminometer 1360, where eachreaction tube 162 receives 200 μL of the Detect I reagent followed, byabout a 2 second delay, 200 μL of the Detect II reagent. The velocity atwhich the Detect I and II reagents are injected to the reaction tubes162 is forceful enough to mix the contents of the reaction tubes withoutagitation, and the delivery lines (not shown) are primed so that theflow of reagents is uninterrupted by bubbles or air gaps. The preferredtop velocity for injecting the Detect I and II reagents into thereaction tubes 162 is 1000 μL/sec. As discussed above, the Detect I andII reagents combine to form a basic hydrogen peroxide solution thatenhances the chemiluminescence of those acridinium ester labels whichhave not been hydrolyzed in the selection process. These reagents aresold as the GEN-PROBE® Detection Reagent Kit (Gen-Probe; Cat. No. 1791).

In step 1964, the MTU 160 passes from the luminometer 1360 to thedeactivation queue 750. In the deactivation queue 750, 2 mL of ableach-based agent are provided to each of the reaction tubes 162 todeactivate nucleic acid present in the reaction tubes. See, e.g.,Dattagupta et al., U.S. Pat. No. 5,612,200, and Nelson et al., U.S.Patent Application Publication No. US 2005-0202491 A1.

Fluorescence measurements are preferably made at a rate of onemeasurement for each of the three or four spectral bands (i.e., for eachtarget dye) per reaction tube 162 every 30 seconds, and thus thecarousel 1656 must rotate once every 30 seconds. In a preferredembodiment, there are 15 MTUs 160 carried on the carrousel 1656 of theRT incubator 608, each separated by 24°. To permit a read at each of the15 MTU stations 1663 during the rotation, each read must be completed in2 second or less. Ideally, the reads are completed in less than twoseconds, and each rotation is completed in less than 30 seconds toafford time to place new MTUs 160 into the RT incubator 608 as completedMTUs are being removed from the RT incubator 608, while stillmaintaining the desired read rate of one measurement for each dye every30 seconds.

Once the data has been collected by measuring fluorometric emissionsfrom each reaction tube 162 at prescribed intervals for a prescribedperiod of time, the data is processed to determine the concentration ofa particular analyte (e.g., target nucleic acid) in the sample. Themeasured data, that is, the measure signal, will be referred to in termsof a Relative Fluorescent Unit (“RFU”), which is the signal generated bythe printed circuit board 1790 of the optical detection unit 1700 basedon the amount of emission fluorescence focused onto the photo diode1780. Each data point, measured at a given time interval, is RFU(t).Plots of RFU(t) for a variety of data sets, known as “growth curves” areshown in FIG. 78. In general, each RFU(t) plot is generally sigmoidal inshape, characterized by an initial, flat portion (known as the “staticlevel” or “baseline phase”) at or near a minimum level, followed by anabrupt and relatively steeply sloped portion (known as the “growthphase”), and ending with a generally flat portion at or near a maximumlevel (known as the “plateau phase”).

As used herein, a “growth curve” refers to the characteristic pattern ofappearance of a synthetic product, such as an amplicon, in a reaction asa function of time or cycle number. A growth curve is convenientlyrepresented as a two-dimensional plot of time (x-axis) against someindicator of product amount, such as a fluorescence measurement—RFU(y-axis). Some, but not all, growth curves have a sigmoid-shape. The“baseline phase” of a growth curve refers to the initial phase of thecurve wherein the amount of product (such as an amplicon) increases at asubstantially constant rate, this rate being less than the rate ofincrease characteristic of the growth phase (which may have a log-linearprofile) of the growth curve. The baseline phase of a growth curvetypically has a very shallow slope, frequently approximating zero. The“growth phase” of a growth curve refers to the portion of the curvewherein the measurable product substantially increases with time.Transition from the baseline phase into the growth phase in a typicalnucleic acid amplification reaction is characterized by the appearanceof amplicon at a rate that increases with time. Transition from thegrowth phase to the plateau phase of the growth curve begins at aninflection point where the rate of amplicon appearance begins todecrease. The “plateau phase” refers to the final phase of the curve. Inthe plateau phase, the rate of measurable product formation issubstantially lower than the rate of amplicon production in thelog-linear growth phase, and may even approach zero.

A process for calculating an analyte concentration is shown by means ofa flow chart in FIG. 77. The data RFU(t) from the optical detectionmodule 1700 is input as represented at box 2100. At step 2102, the dataRFU(t) goes through a color separation procedure. As can be appreciatedfrom FIG. 67, there can be considerable overlap in the emission spectraof different dyes, especially spectrally adjacent dyes. Accordingly, theRFU(t) data obtained from a particular reaction tube 162 may compriseemission data corresponding to the analyte of interest (i.e., from thedye joined to the probe that binds to the analyte of interest) as wellas emission data from one or more different dyes corresponding todifferent targets. To separate that portion of the RFU(t) signal that isnot due to the analyte of interest, standard mathematical techniques,such as deconvolving the different signals obtained by the different,dye-specific optical detection modules 1700, can be employed.Deconvolving is a well known technique in which it is assumed that thesignal measured by each optical detection module can be represented as amathematical function of the emission from each of the dyes present inthe sample. For example, assuming that measurements are made with fouroptical detection modules:RFU ₁(t)=k ₁ RFU _(A)(t)+k ₂ RFU _(B)(t)+k ₃ RFU _(C)(t)+k ₄ RFU _(D)(t)RFU ₂(t)=k ₅ RFU _(A)(t)+k ₆ RFU _(B)(t)+k ₇ RFU _(C)(t)+k ₈ RFU _(D)(t)RFU ₃(t)=k ₉ RFU _(A)(t)+k ₁₀ RFU _(B)(t)+k ₁₁ RFU _(C)(t)+k ₁₂ RFU_(D)(t)RFU ₄(t)=k ₁₃ RFU _(A)(t)+k ₁₄ RFU _(B)(t)+k ₁₅ RFU _(C)(t)+k ₁₆ RFU_(D)(t)

where:

-   -   RFU₁(t)=signal measured at optical detection module #1;    -   RFU₂(t)=signal measured at optical detection module #2;    -   RFU₃(t)=signal measured at optical detection module #3;    -   RFU₄(t)=signal measured at optical detection module #4.    -   RFU_(A)(t), RFU_(B)(t), RFU_(C)(t), RFU_(D)(t)=portion of the        emission signal due to each of dyes A, B, C, D; and    -   k₁-k₁₆=constants.

The functions corresponding to the signals for all the optical detectionmodules are placed in a matrix, and a matrix inversion is performed toderive, for each dye, a mathematical representation of the signal due tothat dye as a function of the signals from each of the optical detectionmodules:RFU _(A)(t)=f ₁(RFU ₁(t),RFU ₂(t),RFU ₃(t),RFU ₄(t))RFU _(B)(t)=f ₂(RFU ₁(t),RFU ₂(t),RFU ₃(t),RFU ₄(t))RFU _(C)(t)=f ₃(RFU ₁(t),RFU ₂(t),RFU ₃(t),RFU ₄(t))RFU _(D)(t)=f ₄(RFU ₁(t),RFU ₂(t),RFU ₃(t),RFU ₄(t)).

From color separation 2102, the data RFU(t) goes to threshold timedetermination, which begins at 2104. Threshold time, or T-time, (alsoknown as time of emergence) refers to the time at which the data RFU(t),normalized as discussed below, reaches a predefined threshold value.Using calibration curves, as will be described in more detail below, theT-time determined for a particular sample can be correlated with ananalyte concentration, thereby indicating the analyte concentration forthe sample. In general, the higher the concentration of the analyte ofinterest, the sooner the T-time.

The first step of the T-time determination procedure is backgroundadjustment and normalization of the data, as represented at box 2106.Background adjustment is performed to subtract that portion of thesignal data RFU(t) that is due to background “noise” from, for example,stray electromagnetic signals from other modules of the instrument 50.That is, the background noise includes that part of the RFU(t) signaldue to sources other than the analyte of interest. Background adjustmentis performed by subtracting a background value “BG” from the data RFU(t)to obtain adjusted data RFU*(t). That is, RFU*(t)=RFU(t)−BG.

The background BG can be determined in a number of ways.

In accordance with one method for determining the background noise, thefirst step is to determine the time intervals between data points. Thetime interval is determined by multiplying cycle time (i.e., the timebetween consecutive data measurements) by the data point (i.e., 0^(th)data point, 1^(st) data point, 2^(nd) data point, . . . , n^(th) datapoint) and divide by 60 seconds. For example, assuming a cycle time of30 seconds, the time interval for the 15^(th) data point is (15×30sec.)/60 sec.=7.5.

The next step is to find the midpoint of the signal data by adding theminimum signal data point and the maximum signal data point and dividingby two. That is: (RFU_(max)+RFU_(min))/2 Starting at the timecorresponding to the midpoint value and working backwards, calculate theslope for each pair of data points: (RFU(t)−RFU(t−1))/Δt(t→t−1).

Next, determine where the slope of RFU(t) flattens out by finding thefirst slope value that is less than the static slope value (i.e., thevalue before the RFU(t) curve begins its upward slope). A representativestatic slope value, also known as the “delta value,” includes 0.0001.Once this slope is found, find the next cycle in which the slope that isnot negative or is, for example, above the negative delta value (i.e.,−0.0001); this value is H_(index). Next, take the mean of the entirerange of RFU(t) values starting at the first data point and go to theRFU value that corresponds to the H_(index) value. The mean of this datamay be computed using the Excel TRIMMEAN function on this range of datausing a static back trim value of 0.15 (that is, the lowest 7.5% of RFUvalues in the specified range and the highest 7.5% RFU values in thespecified range are excluded). This mean value is the background, BG.

Alternatively, the background can be determined in accordance with theprocedure described above using a delta value other than 0.0001.

A further alternative method for determining the background eliminatesthe delta value criterion and instead take a TRIMMEAN mean of the RFUdata from cycle 1 to a prescribed end point, such as the first cyclebefore 5.5 minutes. For this alternative, the static back trim value maybe adjusted to, for example, 0.40 (that is, the lowest 20% of RFU valuesin the specified range and the highest 20% RFU values in the specifiedrange are excluded from the background calculation).

A further alternative method for determining the background is toperform a curve fit on all or a portion of the RFU data to derive anestimate of the baseline value, which is the background to besubtracted. Any curve fit technique suitable for fitting a curve to theRFU data can be used.

An exemplary curve fit technique is to use a portion of the equationderived by Weusten et al. for curve fit of the typically sigmoidalcurves associated with nucleic acid amplification. See Weusten et al.,Nucleic Acids Research, 30(6e26): 1-7 (2002). For backgroundsubtraction, it is only necessary to ascertain the baseline level. Thus,it is also only necessary to fit a curve to the first portion of the RFUdata encompassing the baseline, usually toward the beginning of thecurve.

The curve fit may be performed on the RFU(t) data from cycle 1 to thecycle just before 75% of the maximum RFU. The following polynomialequation (3), which, as mentioned above, is a portion of the equationderived by Weusten et al, is used to generate a best fit model of theRFU data:RFU(t)=Y0+a1a2[e ^(a2(t-a3))/(1+e ^(a2(t-a3)))]ln(1+e ^(a2(t-a3)))  (3)

Initial estimates for the variables Y0, a1, a2, and a3, as discussedbelow, are input to the curve-fit equation and an iterative solutionfitting the equation to the RFU data is performed, for example, usingthe SOLVER function of Microsoft EXCEL, to yield the final equation andthe final values for Y0, a1, a2, and a3.

-   -   Y0=is the baseline; an initial value can be RFU(1).    -   a1=relates to the steep portion (growth phase) of the RFU(t)        data; 0.05 can be a suitable initial estimate for a1.    -   a2=relates to the steep portion (growth phase) of the RFU(t)        data; 1.0 can be a suitable initial estimate for a2.    -   a3=relates to the transition between the baseline and the slope        feature; the time, or cycle, at which RFU(t) reaches a value        just before 25% of RFU_(max) is a suitable initial estimate for        a3.

When the final values of Y0, a1, a2, and a3 have been derived, Y0 istreated as the back ground, and is subtracted from the RFU(t) data forwhich the curve fit was performed.

Curve fit equations other than that described above can be used. Forexample, the commercially available TABLECURVE software package (SYSTATSoftware Inc.; Richmond, Calif.) can be used to identify and select anequation that described exemplary real-time nucleic acid amplificationcurves. One such exemplary resulting equation, used for mathematicalmodeling, is given by equation (4):RFU(t)=Y0+b(1−exp(−(t−d*ln(1−2ˆ^((−1/e)))−c)/d))^(ˆe)  (4)Still another exemplary resulting equation is given by equation (5):RFU(t)=Y0+b/(1+exp(−(t−d*ln(2ˆ^((1/e))−1)−c)/d))^(ˆe)  (5)In each case, as described above, the equation can be solved, forexample, using the SOLVER function of Microsoft EXCEL, to yield thefinal equation and the final values for Y0 and the other parameters, andthe solutions yields a Y0 that is the background to be subtracted fromthe RFU(t) data.

To normalize the data, each data point, adjusted for the background, isdivided by the maximum data point, also adjusted for the background.That is: $\begin{matrix}{{{Normalized}\quad{RFU}} = {{RFU}_{n}(t)}} \\{= \frac{{RFU}^{*}(t)}{{RFU}_{\max}^{*}}} \\{= \frac{{{RFU}(t)} - {BG}}{{RFU}_{\max} - {BG}}}\end{matrix}$Thus, the RFU_(n)(t) will be from −1 to 1.

In step 2108, the range of data is calculated by subtractingRFU_(n(min)) from RFU_(n(max)). If the calculated range does not meet orexceed a specified, minimum range (e.g., 0.05), the data is consideredsuspect and of questionable reliability, and, thus, the T-time will notbe calculated. The minimum range is determined empirically and may varyfrom one fluorescence measuring instrument to the next. Ideally, thespecified minimum range is selected to ensure that the variation of datavalues from minimum to maximum exceeds the noise of the system. In step2110, a curve fit procedure is applied to the normalized,background-adjusted data. Although any of the well-known curve fitmethodologies may be employed, in a preferred embodiment, a linear leastsquares (“LLS”) curve fit is employed. The curve fit is performed foronly a portion of the data between a predetermined low bound and highbound. The ultimate goal, after finding the curve which fits the data,is to find the time corresponding to the point at which the curveintersects a predefined threshold value. In the preferred embodiment,the threshold for normalized data is 0.11. The high and low bounds aredetermined empirically as that range over which curves fit to a varietyof control data sets exhibit the least variability in the timeassociated with the given threshold value. In the preferred embodiment,the lowbound is 0.04 and the highbound is 0.36. The curve is fit fordata extending from the first data point below the low bound through thefirst data point past the high bound.

At step 2110, determine whether the slope of the fit is statisticallysignificant. For example, if the p value of the first order coefficientis less than 0.05, the fit is considered significant, and processingcontinues. If not, processing stops. Alternatively, the validity of thedata can be determined by the R² value.

The slope m and intercept b of the linear curve y=m×+b are determinedfor the fitted curve. With that information, T-time can be determined atstep 2104 as follows: ${T\text{-}{time}} = \frac{{Threshold} - b}{m}$The technique of using the fitted curve to determine T-times isillustrated graphically in FIG. 79.

Returning to FIG. 77, at step 2116, it is determined whether or notinternal control/calibrator adjustments are desired. Typically, a testprocedure would include at least one reaction vessel with a knownconcentration of a nucleic acid (other than a nucleic acid of interest)as a control, or, alternatively, a control nucleic acid sequence can beadded to each sample. The known concentration can be simply used ascontrol to confirm that a reaction did take place in the reactionvessel. That is, if the known concentration is amplified as expected,successful reaction is confirmed and a negative result with respect tothe target analyte is concluded to be due to absence of target in thesample. On the other hand, failure to amplify the known concentration asexpected indicates a failure of the reaction and any result with respectto the target is ignored.

The known concentration can be used to calibrate the concentration ofthe target. The T-times corresponding to a series of standardscontaining internal control and target sequences are determined for astatistically valid number of data sets. Using this data, a calibrationplot is constructed from which the test sample's concentration isinterpolated as described below.

One method of constructing the calibration plot places the knownconcentrations of target analyte on the x-axis versus the differencebetween target and control T-times on the y-axis. Subsequently, the testsample's concentration is interpolated from the calibration curve fit.Another method of construct the calibration plot places the knownconcentration of target analyte on the x-axis versus the fraction[target T-time/internal control T-time] on the y-axis. Subsequently, thetest sample's concentration is interpolated from the calibration curvefit. An example of this is disclosed in Haaland, et al., “Methods,Apparatus and Computer Program Products for Determining Quantities ofNucleic Acid Sequences in Samples Using Standard Curves andAmplification Ratio Estimates,” U.S. Pat. No. 6,066,458. A furtheralternative method of constructing the calibration plot utilizes aparametric calibration method, such as the method described in Carricket al., “Parametric Calibration Method,” U.S. Provisional ApplicationNo. 60/737,334, which enjoys common ownership herewith.

Occasionally, data sets exhibit a dip just after the initial staticbaseline (i.e., the initial, flat part of the RFU(t) curve, see FIG. 78)and just before the data begins its upward slope. To identify andcorrect such data, and prior to determining the T-time for that data,the following algorithm is employed. Starting at H_(index), check eachRFU(t) value to determine if it is less than the background value, BG.If yes, subtract RFU(t) from BG (the result should be a positivenumber). This will be the CorValue. Add the CorValue to the backgroundsubtracted value, this in turn will bring RFU(t) up to the baseline.Perform this analysis working forward on each RFU(t) value until thelatest CorValue is less than the preceding CorValue. Add the greatestCorValue to each of the remaining background subtracted RFU(t) values.Now, the corrected data set can be normalized and the T-time determinedas described above.

If a curve fit method is used to derive the background level, it may notbe necessary to perform the dip correction described above.

It may also be desirable to perform outlier detection on the data set toidentify and, if necessary, discard data points that exhibit abnormalvalues as compared to the remaining data points. Any of the well-knownoutlier detection methodologies can be used.

The quantitation procedure 2120 is the second part of the analyteconcentration determination. T-times are determined for knownconcentrations of analytes for known conditions. Using this data,relationships between analyte concentrations (typically expressed as logcopy) and T-times can be derived. After a T-time is determined for aparticular sample, the derived relationship (Log copy=f (T-time)) can beused to determine the analyte concentration for the sample.

More specifically, at steps 2122 and 2124, calibration/control data setsfor a control analyte of known concentrations are validated by, forexample, outlier analysis and/or any other known data validationmethodologies. If the data is found to be valid, calibration continues,otherwise, calibration stops.

T-times for the control data sets are determined, and T-time vs. Logcopy is plotted for all samples of a particular condition (e.g., samplesprocessed with reagents from a particular batch lot). In step 2126, acurve fit, such as a linear least squares fit, is performed on a portionof the T-time vs. Log copy plot to find the slope m and intercept b ofthe line that best fits the data. If the number of available T-time vs.Log copy data points (known as “calibrators”) is not less than apredefined minimum number of calibrators (as determined at step 2128),lowest calibrators, if any, are removed at step 2130, as follows:

After finding the best fit line for the calibrator data points, 2^(nd)and 3^(rd) order curve fits are tested as well. If these fits aresignificantly better than the 1^(st) order, linear fit, the calibratordata point that is furthest from the linear curve fit is discarded, and1^(st), 2^(nd), and 3^(rd) fits are found and compared again with theremaining calibrators. This process is repeated—assuming that the numberof calibrators is not less than the minimum acceptable number ofcalibrators—until the 2^(nd) and 3^(rd) order fits are not significantlybetter than the 1^(st) order, linear fit.

When the linear T-time vs. Log copy equation has been derived, theconcentration (as Log copy) of the analyte of interest for a sample isdetermined, at step 2132, by plugging the T-time for that sample intothe equation. Thus, the assay results are obtained 2134.

Possible enhancements of the RT incubator 608 include self checkingoptical detection modules. In such a module, a known, standardexcitation signal is emitted by the LED 1732 (or, alternatively, aseparate, dedicated LED) and the excitation light is directed to thephoto diode 1780 (and/or a separate, dedicated comparator photo diode)to ensure that the excitation signals, emission signals, and the signaloutput of the printed circuit board 1790 are all correct.

All documents referred to herein are hereby incorporated by referenceherein. No document, however, is admitted to be prior art to the claimedsubject matter.

While the invention has been described in connection with what arepresently considered to be the most practical and preferred embodiments,it is to be understood that the invention is not to be limited to thedisclosed embodiments, but, on the contrary, is intended to covervarious modifications and equivalent arrangements included within thespirit and scope of the appended claims.

Furthermore, those of the appended claims which do not include languagein the “means for performing a specified function” format permittedunder 35 U.S.C. § 112(¶6), are not intended to be interpreted under 35U.S.C. §112(¶6) as being limited to the structure, material, or actsdescribed in the present specification and their equivalents.

1. A method for determining the amount of an analyte of interest in asample, the method comprising: (a) collecting data comprising periodicmeasurements of the level of a signal emitted by the sample, the levelof the signal being associated with the presence of the analyte ofinterest in the sample; (b) adjusting the data collected in step (a) toremove background contributions to the measured levels associated withsources other than the presence of the analyte of interest; (c)normalizing the data adjusted in step (b) by dividing the adjusted databy the maximum level measured, adjusted to remove backgroundcontributions; (d) approximating a curve through a portion of theadjusted, normalized data between a predetermined low bound andpredetermined high bound; (e) determining an emergence time at which thecurve approximated in step (d) crosses a predetermined threshold datalevel; and (f) determining the amount of the analyte of interest bycomparing the emergence time determined in step (e) with threshold timesdetermined for known amounts of the analyte of interest.
 2. The methodof claim 1, wherein the approximating step comprises applying a curvefit procedure to the adjusted, normalized data points between thepredetermined low bound and predetermined high bound.
 3. The method ofclaim 2, wherein the curve fit procedure is a linear least squares fit.4. The method of claim 1, wherein the data collected comprisesmeasurements of the level of the signal emitted taken at least onceevery 30 seconds.
 5. The method of claim 1, wherein the predeterminedlow bound is about 0.04 and the predetermined high bound is about 0.36.6. The method of claim 1, wherein the normalized predetermined thresholddata level is about 0.11.
 7. The method of claim 1, wherein saidadjusting step comprises determining a level of background contributionand then subtracting the determined level of background contributionfrom the data collected in step (a).
 8. The method of claim 7, whereindetermining the level of background contribution comprises a step fordetermining an amount of background contribution.